fluorescence loss after fixation

From:Gayle Callis

You wrote: 
RE:  fluorescein
From:"C.M. van der Loos" 
Would it not be better to trace something that is non-fluorescent but 
certainly survives the fixation procedure? I mean something like 
digoxigenin that is also easy coupled to any protein (like FITC-
coupling. Via an anti-DIG/FITC antibody this hapten can be traced 
again. I believe there is a considerable risk that any fluorochrome can 
damaged by fixation techniques.

Dear All and Chris, 
I thought about this and remembered our immunologists work with directly
labeled antibodies i.e. CD4-FITC,-PE, -Rhodamine, etc, then stain isolated
lymphocytes. AFTER immunofluorescently labeling/staining the cells, they
often fix them with paraformaldehyde overnight, rinse with buffer the next
day, and do FACS analysis without any loss of fluorescence at all. I don't
think fixation is damaging their fluorescent markers and one should be able
to apply this to tissue sections as well. 

I think the one thing people need to be aware of is the pH requirements of
the fluorophore.  Obviously the pH of the fixative would come into play here. 

It is wise to check solvent, pH, temperature, enzyme exposures, detergents,
etc anything than will damage the fluorescent molecule before embarking on
an the experiment.  Molecular Probes (www.probes.com) is the ideal place to
do.  They are THE experts in the fluorescent molecule market with rapid
response to peoples glowing problems. 

One question:  the person asking about fluorescein never said what
application they were using. Maybe they will enlighten us.  It is not
uncommon to work with a live cells or a whole organ, i.e. spleen, lymph
node and watch FITC and other IFA labeled cells traffic through the tissue
using multiphoton laser scanning microscopy. Very elegant work done in real
time! More and more fluorescent bioimaging is being done these days even at
the single molecule level.  

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

email: gcallis@montana.edu

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