Vendor reply: Tyramide superboost kits quantitative?

[Kilgore, Jason A.]

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**Vendor Reply**

 

Some good suggestions, George.

 

I would add that the SuperBoost kits come with a Stop Solution.  For quantitative imaging, it would be crucial to control the timing of the reaction and to use the Stop Solution.  Also, there may be slight differences between lots for the components, such as the enzymatic activity of the HRP.

 

(of course, there’s also the concerns about true quantitation with microscopy, but I’ll defer to Jim Pawley and his “39 steps” article, https://www.ncbi.nlm.nih.gov/pubmed/10818693, and Jennifer Waters’ article http://jcb.rupress.org/content/185/7/1135 )

 

Jason

 

 

Jason A. Kilgore

Technical Application Scientist

Molecular Probes / EVOS Tech Support

Life Sciences Solutions

 

Thermo Fisher Scientific

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Thursday, October 13, 2016 11:24 AM
To: [hidden email]
Subject: Re: Tyramide superboost kits quantitative?

 

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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:

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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, PhD

Houston, TX 77054

[hidden email]

https://www.linkedin.com/in/georgemcnamara

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http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650