Re: fluorescein

From:Gayle Callis

Storage should be in the dark, to prevent photo bleaching of FITC when
samples are collected, fixed, handled - etc.    After cutting sections must
be in dark after mounting with antifade mounting media.  Vector has a nice
one, Vectashield Hardset.  

She can probably do all three preparations, but remember that if she fixes
in formalin or paraformaldehyde, the tissue will have some
autofluorescence.  If the FITC signal is stronger, brighter than then
dimmer autofluoresence, then all should go well.  In general, the
microcapsules are huge! and very bright. 

I would think ingesting FITC might damage it (digestive enzymes, etc) but
not sure, there may be some pH and enzyme considerations.  Have her contact
tech services at Molecular Probes, they usually have answers for

We have used FITC labelled microcapsules injected into bovine tonsil,
removed tissue, snap freeze and cryosectioned and fixed sections
immediately with NBF. Some autofluorescence occurs but avoid acetone, it
damages FITC or rather causes loss of fluorescence. After section NBF
fixation, rinse with PBS, sections were mounted with aqueous mounting media
compatible for fluorescent molecules. What little autofluorescence occured
was dim, plus looking at both red and green channel gave interesting
results, with autofluorescence appearing reddish and microcapsules still
bright yellow-green. It was a nice way to use autofluorescence as contrast.
 So examine sections with both excitations for FITC and Rhodamine, you get
interesting results, and be sure to examine with colocalization for those
interesting results. 

Wouldn't it be cool if she could examine these tiny fish live with
multiphoton confocal laser scanning microscope - she could track the
capsules in real time whole they are ingesting the capsules!   


At 11:40 AM 7/17/2003 +0200, you wrote:
>Dear List-Members
>Not really an Histology question, but related. A colleague, who is working
with diets for fish larvae wants to put fluorescein (FITC)as a marker in
the food (microcapsules) for fish larvae, and after to measure the
fluorescence. She would like to store the samples in some way until the
measurements of fluorescence. The question is, does fluorescein would be
afected by the storing of the sample? (she said to me she could freeze,
freeze-dry, or maybe fix, in this last case, what would be the best
fixative?). I have never worked with fluorescence, so I decided to ask the
experts. Any advice will be welcome.
>thanks in advance 
>Jos#233# Luis
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)


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