Cy7-TSA dye

[Sripad Ram-2]

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Hello,
Just wondering if anyone has ever come across (or used) a Cy7 or similar
TSA dye probe? I checked a few of the vendors that I could think of
(Molecular Probes/Invitrogen/Fisher, Perkin Elmer) but none of them had any
probes in that range.

The goal is to do automated staining of FFPE tissues using Cy5 and another
NIR probe. The dyes need to be TSA conjugates. I do not want to use Cy5.5
due to spectral overlap. Also, dyes in the visible region are no beuno due
to too much autofluorescence.

Any help/suggestion is greatly appreciated.

Thanks.

Sripad

[George McNamara]

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Hi Sripad,

Molecular Probes can conjugate whatever Alexa Fluor you want onto
tyramide, and yes, there is an AF Cy7 equivalent.Tyramide is off patent,
other 'chemistry' companies could conjugate their/your favorite
fluorophores.

//

At higher plex, you are going to have spectral overlap: learn to love
spectral unmixing. For example:

PLoS One. 2016 Jul 8;11(7):e0158495. doi: 10.1371/journal.pone.0158495.
eCollection 2016.

Multiplexed Spectral Imaging of 120 Different Fluorescent Labels.

Valm AM(1)(2), Oldenbourg R(1)(2), Borisy GG(3).

Author information:
(1)Marine Biological Laboratory, Woods Hole, Massachusetts, United States of
America.
(2)Brown University, Providence, Rhode Island, United States of America.
(3)Forsyth Institute, Cambridge, Massachusetts, United States of America.

The number of fluorescent labels that can unambiguously be distinguished in a
single image when acquired through band pass filters is severely limited by the
spectral overlap of available fluorophores. The recent development of spectral
microscopy and the application of linear unmixing algorithms to spectrally
recorded image data have allowed simultaneous imaging of fluorophores with highly
overlapping spectra. However, the number of distinguishable fluorophores is still
limited by the unavoidable decrease in signal to noise ratio when fluorescence
signals are fractionated over multiple wavelength bins. Here we present a
spectral image analysis algorithm to greatly expand the number of distinguishable
objects labeled with binary combinations of fluorophores. Our algorithm utilizes
a priori knowledge about labeled specimens and imposes a binary label constraint
on the unmixing solution. We have applied our labeling and analysis strategy to
identify microbes labeled by fluorescence in situ hybridization and here
demonstrate the ability to distinguish 120 differently labeled microbes in a
single image.

DOI: 10.1371/journal.pone.0158495
PMCID: PMC4938436
PMID: 27391327



George


On 7/28/2017 2:22 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.
> *****
>
> Hello,
> Just wondering if anyone has ever come across (or used) a Cy7 or similar
> TSA dye probe? I checked a few of the vendors that I could think of
> (Molecular Probes/Invitrogen/Fisher, Perkin Elmer) but none of them had any
> probes in that range.
>
> The goal is to do automated staining of FFPE tissues using Cy5 and another
> NIR probe. The dyes need to be TSA conjugates. I do not want to use Cy5.5
> due to spectral overlap. Also, dyes in the visible region are no beuno due
> to too much autofluorescence.
>
> Any help/suggestion is greatly appreciated.
>
> Thanks.
>
> Sripad

--


George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu (as of May 22, 2017)

[George McNamara]

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*****

Hi again Sripad,

a do-it-yourself conjugation method of NHS-fluorophore and tyramine is in:

Each GPI-anchored protein species forms a specific lipid raft depending
on its GPI attachment signal.

Miyagawa-Yamaguchi A,*Kotani N*,*Honke*K.

Glycoconj J. 2015 Oct;32(7):531-40. doi: 10.1007/s10719-015-9595-5. Epub
2015 May 7.

PMID:
    25948169


We previously reported a method, termed enzyme-mediated activation of
radical sources (EMARS) for analysis of co-clustered molecules with
horseradish peroxidase (HRP) fusion proteins expressed in living cells.
This method is featured by radical formation of*labeling*reagents by
HRP. In the current study, we have employed another*labeling*reagent,
fluorescein-conjugated*tyramide*(FT) instead of the original arylazide
compounds. Although hydrogen peroxide is required for the activation of
FT, the*labeling*efficiency by HRP and the nonspecific reactions by
endogenous enzyme(s) have been dramatically improved compared with the
original fluorescein arylazide. This revised EMARS method has enabled
visualization of co-clustered molecules in the endoplasmic reticulum and
Golgi membranes with confocal microscopy. By using this method, we have
found that GPI-anchored proteins, decay accelerating factor (DAF) and
Thy-1 are exclusively co-clustered with HRP-DAFGPI and HRP-Thy1GPI, in
which GPI attachment signals of DAF and Thy-1 have been connected to
HRP, respectively. Furthermore, the N-glycosylation types of DAF and
Thy-1 have been found to correspond to those of HRP-DAFGPI and
HRP-Thy1GPI, respectively. These results indicate that each GPI-anchored
protein species forms a specific lipid raft depending on its GPI
attachment signal, and that the EMARS method can segregate individual
lipid rafts.


        KEYWORDS:

EMARS; Fluorescein*tyramide*; HRP; N-glycosylation; Visualization of
co-clustering molecules

PMID:
    25948169   DOI: 10.1007/s10719-015-9595-5



Synthesis of fluorescein-conjugated tyramide

100 mg of tyramine (Sigma) dissolved in 5 mL N,Ndimethylacetamide (DMAA)
was added to 100 mg NHS fluorescein (Thermo) in 5 mL DMAA. The mixture
was incubated at roomtemperature for 12 h in dark. An aliquot of the
mixture was applied to thin-layer chromatography
(chloroform:methanol:water = 65:25:4 vol/vol) to check whether
NHS-fluorescein was completely consumed. To remove excess tyramine, the
mixture was applied to 8 mL of cation-exchange resin, AG-50WX8 (Bio-Rad)
equilibrated with DMAA. An aliquot of the flow-through fraction was
applied to thin-layer chromatography again to check whether tyramine was
completely removed. The flow-through fraction containing
fluorescein-conjugated tyramine (FT) was stored at −20 °C.


//

Alexa Fluor 750 is the closest Alexa to Cy7 (see Semrock Searchlight or
Chroma Spectra Viewer for spectra). Many companies now offer NIR
fluorophores.

Alternatively, you could buy biotin-tyramide and AF750-streptavidin or
Cy7-streptavidin. Other indirect detection schemes exist, such as
digoxigenin-tyramide (find online or make your own) and
fluorophore-anti-DIG.

enjoy,

George



On 7/29/2017 10:05 AM, George McNamara wrote:

>
> Hi Sripad,
>
> Molecular Probes can conjugate whatever Alexa Fluor you want onto
> tyramide, and yes, there is an AF Cy7 equivalent.Tyramide is off
> patent, other 'chemistry' companies could conjugate their/your
> favorite fluorophores.
>
> //
>
> At higher plex, you are going to have spectral overlap: learn to love
> spectral unmixing. For example:
>
> PLoS One. 2016 Jul 8;11(7):e0158495. doi: 10.1371/journal.pone.0158495.
> eCollection 2016.
>
> Multiplexed Spectral Imaging of 120 Different Fluorescent Labels.
>
> Valm AM(1)(2), Oldenbourg R(1)(2), Borisy GG(3).
>
> Author information:
> (1)Marine Biological Laboratory, Woods Hole, Massachusetts, United States of
> America.
> (2)Brown University, Providence, Rhode Island, United States of America.
> (3)Forsyth Institute, Cambridge, Massachusetts, United States of America.
>
> The number of fluorescent labels that can unambiguously be distinguished in a
> single image when acquired through band pass filters is severely limited by the
> spectral overlap of available fluorophores. The recent development of spectral
> microscopy and the application of linear unmixing algorithms to spectrally
> recorded image data have allowed simultaneous imaging of fluorophores with highly
> overlapping spectra. However, the number of distinguishable fluorophores is still
> limited by the unavoidable decrease in signal to noise ratio when fluorescence
> signals are fractionated over multiple wavelength bins. Here we present a
> spectral image analysis algorithm to greatly expand the number of distinguishable
> objects labeled with binary combinations of fluorophores. Our algorithm utilizes
> a priori knowledge about labeled specimens and imposes a binary label constraint
> on the unmixing solution. We have applied our labeling and analysis strategy to
> identify microbes labeled by fluorescence in situ hybridization and here
> demonstrate the ability to distinguish 120 differently labeled microbes in a
> single image.
>
> DOI: 10.1371/journal.pone.0158495
> PMCID: PMC4938436
> PMID: 27391327
>
>
> George
>
>
> On 7/28/2017 2:22 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 onhttp://www.imgur.com  and include the link in your posting.
>> *****
>>
>> Hello,
>> Just wondering if anyone has ever come across (or used) a Cy7 or similar
>> TSA dye probe? I checked a few of the vendors that I could think of
>> (Molecular Probes/Invitrogen/Fisher, Perkin Elmer) but none of them had any
>> probes in that range.
>>
>> The goal is to do automated staining of FFPE tissues using Cy5 and another
>> NIR probe. The dyes need to be TSA conjugates. I do not want to use Cy5.5
>> due to spectral overlap. Also, dyes in the visible region are no beuno due
>> to too much autofluorescence.
>>
>> Any help/suggestion is greatly appreciated.
>>
>> Thanks.
>>
>> Sripad
>
> --
>
>
> George McNamara, PhD
> Baltimore, MD 21231
> [hidden email]
> https://www.linkedin.com/in/georgemcnamara
> https://works.bepress.com/gmcnamara/75    (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
> http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
> http://confocal.jhu.edu  (as of May 22, 2017)
>

--




George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Sripad Ram-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

George,
Thanks for the info. I am aware of the paper that you mentioned. They use a
combinatorial strategy to achieve the high level of multiplexing, which is
something that we cannot translate easily to other applications.

Yes, spectral imaging is the way to go but it has its own challenges. I
have been doing spectral unmixing for a couple of years and part of my goal
to go with a Cy7-TSA probe is to move away from spectral unmixing.

Regards,
Sripad



On Sat, Jul 29, 2017 at 7:05 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 Sripad,
>
> Molecular Probes can conjugate whatever Alexa Fluor you want onto
> tyramide, and yes, there is an AF Cy7 equivalent.Tyramide is off patent,
> other 'chemistry' companies could conjugate their/your favorite
> fluorophores.
>
> //
>
> At higher plex, you are going to have spectral overlap: learn to love
> spectral unmixing. For example:
>
> PLoS One. 2016 Jul 8;11(7):e0158495. doi: 10.1371/journal.pone.0158495.
> eCollection 2016.
>
> Multiplexed Spectral Imaging of 120 Different Fluorescent Labels.
>
> Valm AM(1)(2), Oldenbourg R(1)(2), Borisy GG(3).
>
> Author information:
> (1)Marine Biological Laboratory, Woods Hole, Massachusetts, United States
> of
> America.
> (2)Brown University, Providence, Rhode Island, United States of America.
> (3)Forsyth Institute, Cambridge, Massachusetts, United States of America.
>
> The number of fluorescent labels that can unambiguously be distinguished
> in a
> single image when acquired through band pass filters is severely limited
> by the
> spectral overlap of available fluorophores. The recent development of
> spectral
> microscopy and the application of linear unmixing algorithms to spectrally
> recorded image data have allowed simultaneous imaging of fluorophores with
> highly
> overlapping spectra. However, the number of distinguishable fluorophores
> is still
> limited by the unavoidable decrease in signal to noise ratio when
> fluorescence
> signals are fractionated over multiple wavelength bins. Here we present a
> spectral image analysis algorithm to greatly expand the number of
> distinguishable
> objects labeled with binary combinations of fluorophores. Our algorithm
> utilizes
> a priori knowledge about labeled specimens and imposes a binary label
> constraint
> on the unmixing solution. We have applied our labeling and analysis
> strategy to
> identify microbes labeled by fluorescence in situ hybridization and here
> demonstrate the ability to distinguish 120 differently labeled microbes in
> a
> single image.
>
> DOI: 10.1371/journal.pone.0158495
> PMCID: PMC4938436
> PMID: 27391327
>
>
>
> George
>
>
> On 7/28/2017 2:22 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.
>> *****
>>
>>
>> Hello,
>> Just wondering if anyone has ever come across (or used) a Cy7 or similar
>> TSA dye probe? I checked a few of the vendors that I could think of
>> (Molecular Probes/Invitrogen/Fisher, Perkin Elmer) but none of them had
>> any
>> probes in that range.
>>
>> The goal is to do automated staining of FFPE tissues using Cy5 and another
>> NIR probe. The dyes need to be TSA conjugates. I do not want to use Cy5.5
>> due to spectral overlap. Also, dyes in the visible region are no beuno due
>> to too much autofluorescence.
>>
>> Any help/suggestion is greatly appreciated.
>>
>> Thanks.
>>
>> Sripad
>>
>
> --
>
>
> George McNamara, PhD
> Baltimore, MD 21231
> [hidden email]
> https://www.linkedin.com/in/georgemcnamara
> https://works.bepress.com/gmcnamara/75   (may need to use Microsoft Edge
> or Firefox, rather than Google Chrome)
> http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
> http://confocal.jhu.edu (as of May 22, 2017)
>