Tyramide superboost kits quantitative?

[Claire Brown]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

[George McNamara]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information: 
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning 
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 
PMCID: PMC3777543
PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

[Sripad Ram-2]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

Thanks!

Sripad Ram

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information: 
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning 
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 
PMCID: PMC3777543
PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

[George McNamara]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Sripad,

Phillip Moen is the best I can do for TSA products per se. His linkedin profile is

https://www.linkedin.com/in/phillip-moen-2642184

Tom Deerink, Mark Ellisman, roger Tsien et al have correlative LM-EM for diaminobenzidine from eosin, miniSOG, etc. The DAB monomer is al little smaller than fluorophore-tyramide, likely similar radicals either way.

Now with super-resolution, say collagen 2D matrix, detect with limiting dilution anti-collagen, detect that with HRP- or HRP-polymer antibody or streptavidin, and detect with Alexa Fluor 647-tyramide, add the latest best super-res mounting medium, acquire a couple of images and publish.

Molecular Probes (Thermo Fisher) licensed TSA from PerkinElmer, so the fundamentals are the same. Details in the reagent kits probably do not matter much and will be mostly interchangable.

My brain dump on microscopy through ~2005 is in the MPMicro download at

https://works.bepress.com/gmcnamara/2

FP table a bit out of date, see http://www.geomcnamara.com/fluorescent-proteins-photophysics-data

I haven't looked to see if I had FP biosensors, current table of very basic info is at http://www.geomcnamara.com/fluorescent-biosensors 

FP Biosensors table is currently lacking key references -- these are:

  http://biosensor.dpb.carnegiescience.edu/

  https://www.ncbi.nlm.nih.gov/pubmed/21456512   and their supplemental file at http://pubs.acs.org/doi/suppl/10.1021/cr100002u

At some point I'll post the fluorophores table from my Current Protocols unit, but that is a limited list, and not much different than in the PubSpectra XLSX file inside the ZIP download at

https://works.bepress.com/gmcnamara/9/

George

On 10/13/2016 6:20 PM, S Ram wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

Thanks!

Sripad Ram

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information: 
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning 
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 
PMCID: PMC3777543
PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

[George McNamara]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Dear Confocal Listserv readers,

If any of your favorite fluorescent dyes (small molecules, not fluorescent proteins) are not in the draft table below, please send them to me in the same column format, and I can add them to my draft. The table should appear in my Current Protocols chapter, and sooner online at http://www.geomcnamara.com/data which already has a fluorescent proteins data table (not photo$able FPs) and very basic list of FP biosensors. You are free to repost: data are facts, facts are not copyrightable.

Enjoy,

George

Table 4.4.2 Fluorophores Photophysics Data

Fluorophore	Abs or Ex max (nm)	Em max (nm)	Extinction Coefficient	Quantum Yield	Brightness Index
Acridine orange	271	520	27,000	0.20	5
Alexa Fluor 430	431	541	16,000	0.55	9
Alexa Fluor 488	495	519	71,000	0.94	67
Alexa Fluor 532	532	553	81,000	0.80	65
Alexa Fluor 546	556	573	104,000	0.96	100
Alexa Fluor 568	578	603	91,300	0.75	69
Alexa Fluor 594	590	617	73,000	0.64	47
Allophycocyanin (APC)	650	660	700,000	0.68	476
Atto 390	390	479	24,000	0.90	22
Atto 425	436	484	45,000	0.65	29
Atto 430LS	433	547	32,000	0.65	21
Atto 465	453	508	75,000	0.75	56
Atto 490LS	496	661	40,000	0.30	12
Atto 520	525	547	105,000	0.95	100
Atto 532	534	560	115,000	0.90	104
Atto 565	566	590	120,000	0.97	116
Atto 590	598	634	120,000	0.90	108
Atto 610	616	646	150,000	0.70	105
Atto 620	620	641	120,000	0.50	60
Atto 635	637	660	120,000	0.45	54
Atto 655	655	680	125,000	0.50	63
Atto 680	675	699	125,000	0.40	50
ATTO-Dino 1 (dsDNA)	490	531	179,000	0.70	125
ATTO-Dino 2 (dsDNA)	506	535	162,000	0.70	113
Bacteriochlorin (“BC”)	713	717	120,000	0.11	13
Bacteriochlorin BC-T2-T12	737	744	120,000	0.14	17
Bacteriochlorin BC-Ph3-Ph13	736	742	120,000	0.12	14
Bacteriochlorin BC-T2-OMe5-T12	732	739	120,000	0.18	22
Bacteriochlorin BC-V3-V13	750	756	120,000	0.10	12
Bacteriochlorin BC-PE3-PE13	763	768	120,000	0.15	18
Bacteriochlorin BC-A3-A13	768	774	120,000	0.09	11
Bacteriochlorin BC-F3-F13	771	777	120,000	0.07	8
BODIPY 507/545	513	549	82,800	0.73	60
BODIPY FL	504	510	70,000	0.90	63
BODIPY TR	588	616	68,000	0.84	57
B-phycoerythrin (B-PE)	545	575	2,410,000	0.98	2,362
Calcein	494	516	81,000	0.78	63
Cascade Blue	378	423	26,000	0.54	14
Chromeo 488	488	517	73,000	0.27	20
Chromeo 494	494	628	55,000	0.15	8
Chromeo 505	505	526	70,000	0.30	21
Chromeo 546	545	561	98,800	0.15	15
Chromeo 642	642	660	180,000	0.21	38
Chromeo P429 Py-Dye	429	536	75,000	0.10	8
Chromeo P503 Py-Dye	503	600	24,000	0.50	12
Chromeo P540 Py-Dye	533	627	50,000	0.20	10
Chromeo P543 Py-Dye	543	590	57,000	0.15	9
Coumarin 6	456	500	54,000	0.78	42
Cresyl violet perchlorate	603	622	83,000	0.54	45
Cy3	552	570	150,000	0.15	23
Cy3B	552	570	130,000	0.67	87
Cy5	649	670	250,000	0.28	70
Cy5.5	675	694	250,000	0.23	58
Cy7	755	778	250,000	0.28	70
DAPI (in DMSO)	353	465	27,000	0.58	16
DAPI (in H2O)	344	487	27,000	0.04	1
DsRed	558	583	75,000	0.70	52
Eosin Y	525	543	112,000	0.67	75
EYFP	514	527	84,000	0.61	51
Fluorescein	490	514	90,000	0.92	83
Fluorescein FH3+ (pH <3)	437	-	53,000	-	-
Fluorescein FH2 (pH 4)	434	-	11,000	-	-
Fluorescein FH- (pH 5.3)	472	515	29,000	0.37	11
Fluorescein F2- (pH >8)	490	515	76,900	0.92	71
FM 1-43	479	598	40,000	0.30	12
Fura-2, Ca++ free	363	512	28,000	0.23	6
Fura-2, Ca++ saturated	335	505	34,000	0.49	17
Fura-2, Mn++ saturated	335	-	-	0	0
Fura-2, Zn++ saturated	345	505	34,000	0.69	24
Hoechst 33258 (in DMF)	354	486	46,000	0.35	16
Hoechst 33258 (in H2O)	345	507	46,000	0.03	2
Indo-1, Ca++ free	346	475	33,000	0.38	13
Indo-1, Ca++ saturated	330	401	33,000	0.56	18
IRDye38	778	806	179,000	0.35	62
IRDye40	768	788	140,000	0.38	53
IRDye700	681	712	170,000	0.48	81
IRDye78	768	796	220,000	0.31	68
IRDye80	767	791	250,000	0.21	53
IRDye800	787	812	275,000	0.15	41
JOE	520	548	73,000	0.60	44
Lucifer Yellow CH	230	542	24,200	0.21	5
merocyanine 540	559	579	138,000	0.39	54
neo-Cy5 (DMSO)	656	675	195,000	0.25	49
NIR1	761	796	268,000	0.23	62
NIR2	662	684	250,000	0.34	85
NIR3	750	777	275,000	0.28	77
NIR4	650	671	260,000	0.43	1112
Oregon Green 488	496	516	76,000	0.90	68
Oregon Green 514	506	526	88,000	0.96	85
Oyster® - 645 (ethanol)	651	669	250,000	0.40	100
Oyster® - 656 (ethanol)	665	684	220,000	0.50	11
Pacific Blue	400	447	29,500	0.55	16
Perylene	253	435	38,500	0.94	36
Phenylalanine	222	279	195	0.02	<0.1
POPOP	256	407	47,000	0.93	44
Quinine sulfate (in 0.5M H2SO4)	256	451	5,700	0.55	3
Rhodamine 110	496	520	80,000	0.89	71
Rhodamine 6G	530	552	116,000	0.95	110
Rhodamine B	543	565	106,000	0.70	74
Riboflavin	262	531	34,800	0.37	13
Rose bengal	559	571	90,400	0.11	10
R-Phycoerythrin (R-PE)	480	578	1,960,000	0.68	1,333
SNIR1	666	695	218,000	0.24	52
SNIR3	667	697	245,000	0.24	59
Star 440 SXP	436	515	22,700	0.68	15
Star 470 SXP	472	624	29,000	0.12	4
Star 488	503	524	64,500	0.89	57
Star 512	511	530	84,000	0.82	69
Star 520SXP	515	612	60,000	0.05	3
Star 580	587	607	72,000	0.90	65
Star 600	604	627	43,500	0.73	32
Star 635	639	654	63,000	0.51	32
Star 635P	635	651	125,000	0.92	115
Star Red	638	655	212,000	0.90	191
Sulforhodamine 101	576	591	139,000	0.90	125
Texas Red	586	605	108,000	0.77	83
Texas Red-X	583	603	116,000	0.90	104
TMR	540	565	95,000	0.68	65
Trp	287	348	6,000	0.31	2
Tyr	275	303	1,500	0.21	0.3
Brilliant Violets, Brilliant Ultraviolets, Brilliant Blue
Brilliant Violet BV421	405	421	2,500,000	0.65	1,625
Brilliant Violet BV510	405	510	577,000	0.44	254
Brilliant Violet BV570	405	570	2,300,000	0.08	184
Brilliant Violet BV605	405	603	2,400,000	0.29	696
Brilliant Violet BV650	405	645	2,500,000	0.17	425
Brilliant Violet BV711	405	711	2,800,000	0.15	420
Brilliant Violet BV785	405	785	2,500,000	0.04	100
Brilliant Ultraviolet BUV395	348	395
Brilliant Ultraviolet BUV496	348	496
Brilliant Ultraviolet BUV563	348	563
Brilliant Ultraviolet BUV661	348	661
Brilliant Ultraviolet BUV737	348	737
Brilliant Ultraviolet BUV805	348	805
Brilliant Blue BB515	490	515
Fluorescent nanocrystal quantum dots
Product	Em Peak	Em FWHM	Extinction Coefficient	Quantum Yield	Brightness Index
QD525	525	≤32	320,000	0.60	192
QD565	565	≤34	1,100,000	0.40	440
QD585	585	≤34	2,200,000	0.40	880
QD605	605	≤27	2,400,000	0.40	960
QD625	625				>2,300
QD655	655	≤34	5,700,000	0.40	2,280
QD705	705	wide
eVolve 605	605
eVolve 655	655

Brightness Index = Extinction coefficient * Quantum Yield / 1000. Table features fluorophores for which all four major photophysical parameters, absorption (excitation) maximum, fluorescence emission maximum extinction coefficient (M^-1 cm^-1) and fluorescence quantum yield, are available. QD625 values details have not been published, but is brighter than QD605 or QD655.

Antibody, streptavidin, tetramers/pentamers/dextramers, and other macromolecular labeling have “degree of labeling’ (doL) issues. As a simplistic rule of thumb, one fluorophore can be conjugated to the surface of a protein for every 25,000 dalton molecular weight. An IgG molecule is ~155,000 dalton, so ~6 fluorophores can be conjugated without too much quenching or causing the protein to crash out of solution. With random labeling of lysines, such as isothiocyantate chemistry (FITC, TRITC), there is a risk of the reactive dye making a covalent bond in an Fab binding site, which would occlude binding to the target antigen epitope. If both Fabs were occluded, this dye-IgG would not bind antigenic epitope at all, but could bind non-specifically, increasing background. If there are 50 reactive lysines on the surface of an IgG molecule, and degree of labeling is 6 (+/- some variability), the population of antibody molecules applied to a specimen is going to be very heterogeneous: a small reaction may not have any identical antibody molecules. DoL of different batches of antibodies are unlikely to be identical. Isotype controls, whether a mouse IgG1 monoclonal antibody (defined specificity different than your interest), or polyclonal affinity purified from sera, are at best imperfect controls. Immunoglobulins also vary in glycosylation, disulfide bonding, and single nucleotide polymorphisms – including some that result in amino acid substitutions that affect Fc Receptor binding affinity. A recent (2015) trend has been for vendors, ex. Miltenyi Biotec REAfinity, to use the same “backbone’ for their entire product line antibodies.

The Fluorophore dyes section of the table is abridged from a 4700+ entry data table Excel file the authors posted on the internet,

https://works.bepress.com/gmcnamara/9  = PubSpectra.

the website download also contains 400+ entry fluorescent proteins data table Excel file. Spectra for many of these dyes, and commercial filters and light sources, are available through an interactive website at

http://www.spectra.arizona.edu/

QD### data from Quantum Dot Corp (acquired by Molecular Probes / Thermo Fisher). eVolve 605 and eVolve 655 are Cd containing quantum dots from Affymetrix / eBiosciences (acquired by Thermo Fisher). Bacteriochlorins (BC’s) have small Stokes shifts (Emission maximum – Excitation maximum). They have almost as large extinction coefficients in the ultraviolet, 350-390 nm range, so for practical use have a huge, practical Stokes shifts on the order of 400 nm (they also have an additional excitation peak in the 480-550 nm range. Taniguchi et al (2008) include a graph comparing the full width half maxima (FWHM) emission spectra of Cyanine dyes (~50 nm), near infrared quantum dots (66 nm QD705, 74 nm QD800), BC’s (~20 nm), and ‘expanded’ porphyrins (>120 nm). We suggest with the right excitation light sources (355 nm laser or ~360 and ~390 nm LEDs), and emission separation (filter wheel one camera, cascading filters to multiple cameras or PMTs), that four or more BC’s could be 700-800 nm emission range. If a palette of five BC’s could be closely conjugated to Brilliant Blue BB515, excitation peak 488 nm, extremely efficient energy transfer in the visible absorption peaks (~500-550 nm) could enable extreme multiplexing. Additional multiplexing may be gained by 355 nm excitation of BUV395 to five BC’s. Our thanks to Dr. Bruce Pitner, NIRvana Sciences, for discussions extinction coefficient estimates with respect to NIRvana dyes and Taniguchi et al (2008) and Taniguchi and Lindsey (2016).. Additional dye data from Sednev et al (2015, BioLegend, 2016, Brilliant Violets, http://www.biolegend.com/brilliantviolet 

You can look for updates at at www.GeoMcNamara.com.

On 10/13/2016 6:20 PM, S Ram wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

Thanks!

Sripad Ram

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information: 
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning 
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 
PMCID: PMC3777543
PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

[Mark Cannell-2]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi

This contains some references that may be relevant to the question of how far radicals move?

doi: 10.1177/31.7.6304184J Histochem Cytochem July 1983 vol. 31 no. 7 945-951

HTH Mark

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

Thanks!

Sripad Ram

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information: 
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning 
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 
PMCID: PMC3777543
PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

Mark  B. Cannell Ph.D. FRSNZ FISHR
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[George McNamara]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Mark,

thanks for the find! the kidney GBM laminae rarae's ~60 nm periodicity could make this a nice nanoscopic calibration standard.

I also realized -- from their mention of 2 minute incubation times to limit the DAB + H2O2 reaction -- that my "Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea below could be greatly simplified: detect with tyramide color 1 briefly, then color 2, then color 3, etc.

George

On 10/14/2016 3:07 AM, Mark Cannell wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi

This contains some references that may be relevant to the question of how far radicals move?

doi: 10.1177/31.7.6304184J Histochem Cytochem July 1983 vol. 31 no. 7 945-951

HTH Mark

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

Thanks!

Sripad Ram

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information: 
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning 
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 
PMCID: PMC3777543
PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

Mark  B. Cannell Ph.D. FRSNZ FISHR Professor of Cardiac Cell Biology School of Physiology &  Pharmacology Faculty of Biomedical Sciences University of Bristol Bristol BS8 1TD UK

[hidden email]

[Sripad Ram-2]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Mark and George,

Thanks for the feedback and comments! 

Sripad

On Fri, Oct 14, 2016 at 5:40 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Mark,

thanks for the find! the kidney GBM laminae rarae's ~60 nm periodicity could make this a nice nanoscopic calibration standard.

I also realized -- from their mention of 2 minute incubation times to limit the DAB + H2O2 reaction -- that my "Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea below could be greatly simplified: detect with tyramide color 1 briefly, then color 2, then color 3, etc.

George

On 10/14/2016 3:07 AM, Mark Cannell wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi

This contains some references that may be relevant to the question of how far radicals move?

doi: 10.1177/31.7.6304184J Histochem Cytochem July 1983 vol. 31 no. 7 945-951

HTH Mark

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi George,

As always super informative. Is it just me or do others also think that its high time someone invents a device download all that info that's sitting in your head...?

Very interesting comment about the diffusion radius of the activated TSA radical being less than the diffraction limit. I think that is a very important parameter. Just wondering if you know a reference for that? 

Perkin Elmer recently showed some correlative fluorescence and EM data which suggests similar range of specificity for their TSA staining. 

Thanks!

Sripad Ram

On Thu, Oct 13, 2016 at 11:24 AM, George McNamara <[hidden email]> wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Claire,

Depending on temperature, constant reaction time may be bad. Minimal issue is always run on a warm plate (that is perfectly uniform temperature ... there are slide thermometers for this), incubators or ovens.

I suspect the TSA reaction runs to completion usually, with one of these running out:

* active HRP enzyme molecules (self destructs with some probability for every H2O2 catalytic cycle)

* tyrosine "docking sites" ... both in cells/tissues, the antibody molecules, and the HRPs (an advantage for poly HRP)

* fluorescent tyramide (least likely?)

* H2O2 (also unlikely?)

I believe PerkinElmer's Opal kits for multiplexing deliberately strips the detection Ab-polyHRP, before the next cycle, but have not been able to get data from PerkinElmer on this (and did not need TSA while I worked in a research lab here in Houston).

Many IHC reagent companies sell HRP polymers conjugated to streptavidin or secondary antibodies. For example
STREPTAVIDIN POLY-HRP80 CONJUGATE (plus stabilizer diluent)
https://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html

Yes, you can replace components in the kit with other reagents ... and conversely, use components in the kit for other purposes. An interesting "head to head" is fluorescent tyramide vs DAB IHC, or whatever favorite HRP substrate detection reagent your friendly neighborhood pathologists like.

I'm a big fan of TSA ever  since Phil Moen of NEN (acquired by PerkinElmer) spoke about and demo'd it at a Cold Spring Harbor fluorescence in situ/immuno courses. Phil mentioned the diffusion radius of the activated tyramide radical is less than the diffraction limit of confocal microscopy (i.e. 100 nm radius, 200 nm diameter).

One potential limitation on quantitation: zero reactable tyrosines implies zero signal ... as I mentioned above, both the Ab and HRPs have tyrosines (some even on the surface if I recall correctly).

Big benefit: when done right, can dilute the primary antibody 100 fold (maybe more ... try it), saving money. In fact, failing to do so will result in massive background.

Automation: in principle, automating TSA should improve reproducibility (assuming constant temperature for the reaction, and either constant enzyme reaction time or "run to completion"). However, not paying attention to the cost of the reagents (or stability or lack thereof) could result in one well, $100 in reagents --> one slide).

One of my U Miami customers published a super-duper brightness CD4 and CD8 2 color T-cells (also CD4 and FoxP3) paper ... they killed off the HRP molecules (and before that, endogenous peroxidases) with a terrific product (both great name and works well): PeroxAbolish   http://biocare.net/product/peroxabolish/

https://www.ncbi.nlm.nih.gov/pubmed/21929847

Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub 2011 Sep 
16.

Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant tissue
using laser scanning cytometry.

Takahashi H(1), Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R, Barker
S, Burke GW, Tzakis AG, Ichii H.

Author information: 
(1)Miami Transplant Institute, University of Miami Leonard M. Miller School of
Medicine, Miami, FL, USA.

There is abundant evidence that immune cells infiltrating into a transplanted
organ play a critical role for destructive inflammatory or regulatory immune
reactions. Quantitative in situ analysis (i.e., in tissue sections) of immune
cells remains challenging due to a lack of objective methodology. Laser scanning 
cytometry (LSC) is an imaging-based methodology that performs quantitative
measurements on fluorescently and/ or chromatically stained tissue or cellular
specimens at a single-cell level. In this study, we have developed a novel
objective method for analysis of immune cells, including Foxp3(+) T regulatory
cells (Tregs), on formalin-fixed /paraffin-embedded (FFPE) transplant biopsy
sections using iCys® Research Imaging Cytometer. The development of multiple
immunofluorescent staining was established using FFPE human tonsil sample. The
CD4/CD8 ratio and the population of Tregs among CD4(+) cells were analyzed using 
iCys and compared with the results from conventional flow cytometry analysis
(FCM). Our multiple immunofluorescent staining techniques allow obtaining clear
staining on FFPE sections. The CD4/CD8 ratio analyzed by iCys was concordant with
those obtained by FCM. This method was also applicable for liver, small
intestine, kidney, pancreas, and heart transplant biopsy sections and provide an 
objective quantification of Tregs within the grafts.

DOI: 10.3727/096368911X586747 
PMCID: PMC3777543
PMID: 21929847  [PubMed - indexed for MEDLINE]

and my 2010 book chapter (Yuste 2010 CSHL Press chapter 15 - Imaging in Neurosciences) has a protocol (I would use Mol Probes/ThermoFisher or PerkinElmers ... can probably crosslink the Ab-HRPpolymer onto the cells/tissue instead of their strip).

I also want to acknowledge Molecular Probes - specifically Mike Janes and his team - for visiting Miami (several years ago) for a workshop featuring TSA and Bacmam2.0. Hopefully ThermoFisher still lets the Mol Probes folks out of the lab to do similar workshops.

Speculation (spectral-ation - I currently do not work in a lab, so not going to test this anytime soon -- maybe someone from Mol Probes can try it):

Single molecule localization staining ... or "rainbow single antigen counting by TSA" idea (this will be tedious to do by hand! Payoff: count everything):
=>I suggest using 35 mm imaging dish, so that wash steps can be very large volume (2 mL), and expensive reagent steps just the imaging area (7, 10, 14, 20, 28 mm diameter as appropriate for your expt).
1. incubate primary antibody at ~1/10th of saturation.
2. wash (extensively).
3. incubate secondary antibody-polyHRP.
4. wash extensively.
5. detect with Tyramide color #1.
6. kill HRP, i.e. with PeroxAbolish (http://biocare.net/product/peroxabolish/) AND I suggest gently crosslinking the 2ndAb-HRP-polymer into the specimen (see also Expansion Microscopy approaches).
7. OK, may want to image at this point, ideally with a way to refind the same place later.
*** Repeat 1-6 with different color, optionally also #7.
*. Image everything. See Valm et al https://www.ncbi.nlm.nih.gov/pubmed/27391327 for one example of high multiplexing (not tyramides) using all laser lines and detectors on a spectral confocal microscope.

I see in SuperBoost PDF only 7 colors (7 Alexa's)

https://tools.thermofisher.com/content/sfs/manuals/tyramide_superboost_kits_man.pdf

to get to 10 (ore more) could use biotin. A 1998 paper had do-it-yourself tyramide hapten synthesis
http://jhc.sagepub.com/content/46/6/771.full

Sure, could also do this single color, either add the signals, or photobleach. This could be useful on the MilliporeSigma/EMD Millipore/CellASIC ONIX microperfusion platform (since ONIX only has a few reservoirs and they seem to lack interest in making plates and manifolds for use with 2 or more controllers).

Another option for quantitation: wait for Garry P. Nolan (Stanford Univ.) to publish his immuofluorescence alternative to MIBI-ToF, or collaborate with him to use his MIBI-ToF or buy a Fluidigm imaging CyTOF.

enjoy,

George

On 10/13/2016 11:52 AM, Claire Brown, Dr. wrote:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

I wonder if anyone knows if the new ThermoFisher Tyramide SuperBoost kits are quantitative?

 

https://www.thermofisher.com/order/catalog/product/B40915

 

 

I would assume that keeping the reaction conditions identical from sample to sample would be really important in stopping the HRP reaction after a fixed time but with such amplification is there any way relative signals could be quantified?

 

I would love to hear what people think.

 

Sincerely,

 

Claire

 

 

Mark  B. Cannell Ph.D. FRSNZ FISHR Professor of Cardiac Cell Biology School of Physiology &  Pharmacology Faculty of Biomedical Sciences University of Bristol Bristol BS8 1TD UK

[hidden email]