Tandem dye staining problem

[Wade Sigurdson]

I am forwarding the following question from one of the users of our core facility who is attempting multiple antibody staining.

"Has anyone heard of any issues with multiple antibody staining involving the
tandem dye PerCP-Cy5.5, or tandem dyes, in general? 
Specifically, in a multicolor stained slide containing high density epitopes for
antibodies labeled with PerCp-Cy5.5, Pacific Blue and Alexa647, are there
problems with the FRET transfer or steric interference between large tandem
fluorophores when high density epitopes are simultaneously stained?"

Specifically, samples stained with only the direct conjugated PerCP-Cy5.5 work fine, but when combined with the other directly conjugated antibodies, the PerCP-Cy5.5 staining is not present.  I've never seen this problem before and am at a loss to explain what might be happening.

Thanks,
Wade

[Axel Kurt Preuss]

Only you could quench by overloading

Thanks
Sincerely yours

Axel

IMCB-Central Imaging
[phone]

On Feb 11, 2010, at 9:46 PM, "Wade Sigurdson" <[hidden email]<mailto:[hidden email]>> wrote:

I am forwarding the following question from one of the users of our core facility who is attempting multiple antibody staining.

"Has anyone heard of any issues with multiple antibody staining involving the
tandem dye PerCP-Cy5.5, or tandem dyes, in general?
Specifically, in a multicolor stained slide containing high density epitopes for
antibodies labeled with PerCp-Cy5.5, Pacific Blue and Alexa647, are there
problems with the FRET transfer or steric interference between large tandem
fluorophores when high density epitopes are simultaneously stained?"



Specifically, samples stained with only the direct conjugated PerCP-Cy5.5 work fine, but when combined with the other directly conjugated antibodies, the PerCP-Cy5.5 staining is not present.  I've never seen this problem before and am at a loss to explain what might be happening.

Thanks,
Wade

--
Wade J. Sigurdson, Ph.D.
Director, Confocal Microscope and Flow Cytometry Facility

Voice - 716-829-3589
FAX - 716-829-3589
<mailto:[hidden email]>[hidden email]<mailto:[hidden email]>
<http://www.smbs.buffalo.edu/confocal>www.smbs.buffalo.edu/confocal<http://www.smbs.buffalo.edu/confocal>

Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you.