Re: [Histonet] Autofluorescence problem in olfactory epithelia cryosections

From:Gayle Callis

Dear Tobias,

This has been discussed at great length, even recently, on Histonet.  Check=20
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,=20
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,=20
there is an publication found in J of Histochemistry and Cytochemistry that=20
may help you.

It is always possible to do a colored chromogen with ISH instead of FISH if=20
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 4C 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 10m 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)



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