[Histonet] Autofluorescence problem in olfactory epithelia cryosections

[Gayle Callis]

Dear Tobias,

This has been discussed at great length, even recently, on Histonet.  Check 
the Histonet Archives for even more information on this ongoing annoying 
problem.   Also, I am attaching (privately) a review of autofluorescence 
that may help you understand some of what you are experiencing.  The 
article also has a way to block autofluorescence, although I have never 
tried it.  Anytime you fix tissues with paraformaldehyde or even formalin, 
autofluorescence increases due to aldehyde fixation, something that has 
been observed for many years and with many publications.

Some people, as in our lab, often use autofluorescence like a 
"counterstain" or could we say "counterfluorescence" to contrast with the 
fluorophore you are using.  Blocking autofluorescence is difficult at best, 
there is an publication found in J of Histochemistry and Cytochemistry that 
may help you.

It is always possible to do a colored chromogen with ISH instead of FISH if 
this is unsolvable.  You did not say what your fluorophore is, FITC?

Expect an attachment after this message.

At 06:54 AM 12/2/2004, you wrote:
>Hello,
>
>I've got a serious autoflourescence problem when trying to perform
>ISH on mouse head cryosections.
>
>After ISH process I can see a bright red autoflourescence in large
>parts of my tissue even without any antibodies on it.
>First I thought it might be due to blocking solution containing Horse serum,
>but I test blocked my slides and there was almost no autoflourescence at all.
>So were the untreated slide !
>
>Right now, I don't have a single clue which step of my ISH might cause
>the red autofluorescence but it would be great if anyone of you had a good
>advice for me. Please, let me know !
>
>The mouse has been killed, decapitated, parts of the nose were
>removed and fixed in 4%PFA for 3h. After that the tissue was
>incubated at 4°C in 30% succrose until the tissue sank to the
>bottom of the tube and was than shock frozen and embedded in
>mounting medium. Slices are 10µm thick.
>
>Thank you for your help.
>
>-Tobias-
>
>
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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)