[Histonet] Autofluorescence problems in spleen

From:Gayle Callis

Since autofluorescence problems tend to be light greenish/yellowish tints,
the problem is usually fainter than a good strong signal from Alexa 488 and
sometimes fluorescein. One can use a brighter fluorophore (Alexa 488) or
better yet, use the autofluorescence to your advantage and use a
contrasting color fluorophore e.b. Rhodamine X (Jackson ImmunoResearch)
TRITC-aka another rhodamine, Texas Red, or Alexa 546. 

If you are using eGFP, then you will have problems, unless the
autofluorescence masks the GFP locale. In that case a good antiGFP
antibody, then come back with a fluorophore that brightens up what you want
to see. 

Formalin does create more autofluorescence but if you KNOW what is
autofluorescing, you can sort it out better.  There is a wonderful review
on autofluorescence that I have in PDF file, but I cannot attach that to
Histonet.  If you contact me privately, I will send to you.  The
publication deals with GFP, fixatives and autofluorescence, but still
applies to immunofluorescent staining.  Also, go to Clontech website and
look up the manual titled Living Colours, also for GFP, but this tidy
little publication will tell you more details about what causes problems
and how some people deal with it when working with GFP (Green Fluorescent
Protein).    

People have attempted to get rid of autofluorescence, but we have far less
problems with frozen sections to avoid formalin or paraformaldehyde
fixation (the latter two are prefered fixatives for GFP) although the
problem still exists to some extent with acetone or acetone/alcohol fixed
frozen sections.   
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)



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