Re: Cy7-TSA dye **vendor reply**

[Kilgore, Jason A.]

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** Vendor Reply **

Hi, Sripad,

The Alexa Fluor equivalent of Cy7 is Alexa Fluor 750.  

Currently, Thermo Fisher Scientific (parent company of Molecular Probes) does not offer an AF750 tyramide.  Our Superboost TSA kit (our new TSA version with a poly-HRP) only goes up to AF647 (equivalent to Cy5 in wavelength), so, as mentioned by George, you would either need to conjugate your own (using the AF750 succinimidyl ester, catalog A20011) or have us make a custom for you.  

Contact me offline if you are interested in a custom and I can put you in touch with the appropriate group.

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific
 
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of S Ram
Sent: Saturday, July 29, 2017 11:54 PM
To: [hidden email]
Subject: Re: Cy7-TSA dye

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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)
>