Re: [Histonet] Fluorophore adsorption emission and spectral separation

From:Adam Perry

I had an immunofluorescence question that I'd love to get some input on from anyone out there.  I'm trying to colocalize two antigens in rodent brain sections.  One of the antigens is yielding fairly weak staining- it also happens to be the one that I'm visualizing with Alexa Fluor 488, so I'm getting fairly high background autofluorescence- and I think that the staining could be improved by reducing the autofluorescence.  My DAPI staining (ex/em: 350/470) is only stains nuclei with minimal autofluorescence.  They offer Alexa Fluor 405 streptavidin which has a similar excit/emission range, so I thought labeling with the Alexa Fluor 405 would produce a similarly "clean" background as my DAPI labeling.  Also, I already have the filter sets to work with Alexa Fluor 405 (as opposed to going to the other end of the spectrum).  
  Has anyone used Alexa Fluor 405?  What is your impression of tissue autofluorescence in that range, photostability/bleaching of the compounds, comparison of signal using other fluorophores, etc.
  Any input would be greatly appreciated.
  Adam Perry
  Department of Neuroscience
  University of Illinois at Chicago
  Chicago, IL 60612

Gayle Callis  wrote:

Molecular Probes, go to resources on home page for MP, and click on their 
spectraimager. You can play with the fluorophores you need to see (do it 
as antibodies) and see the graphs for separation - for both CLSM and 
fluorescent microscope work.

With our confocal, adjustment can be made to eliminate 
autofluorescence. I also have a review article on autofluorescence, 
written with GFP in mind, but useful for immunofluorescent work also.

If you want the review, I will send via private email.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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