[Histonet] RE: autofluorescence

[Peter Bannister]

Dear Yasushi.
I have experienced the same autofluorescence in brain tissue and it does 
sound like you are indeed seeing blood vessels.
Try incubating the sections in a solution of 1% Sodium Borohydride (NaBH4) 
in 0.1M Phosphate buffer (not PBS) for about 30 minutes. This substance will 
quench autofluorescnce from free aldehyde groups left over from fixation and 
should eradicate the problem.
If you are using sections on slides then the use of salinated (eg vectabond) 
coated slide will help retain section adhesion. If you are using free 
flating sections then remember not to cap the vial during the incubation. 
The reason is that NaBH4 produces a lot of hydrogen gas when in solution and 
the pressure in the vial will build up and eventually blow the cap off and 
stick the sections to the laboratory wall. This is bad.
Also remember that NaBH4 is pretty nasty stuff when in powdered form so take 
all necessary health and safety precautions.
Good Luck.
Peter Bannister.
>
>Message: 17
>Date: Thu, 11 May 2006 11:12:51 -0500
>From: yasushi nakagawa <nakagawa <@t> umn.edu>
>Subject: [Histonet] autofluorescence?
>To: Histonet <@t> lists.utsouthwestern.edu
>Message-ID: <C75EDC93-ACD7-4EEB-BB23-AA954255A498 <@t> umn.edu>
>Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed
>
>Hi,
>
>We are doing immunohistochemistry using Cy2, Cy3 and Cy5-conjugated
>secondary antibodies on 20um-thick cryosections of embryonic mouse
>brains. Especially with Cy2- filter setting, we sometimes see a
>number of auto-fluorescence-like signals within the brain sections.
>They are thin and long, kind of looking like blood vessels. They show
>up even when we do not use Cy2-conjugated secondaries. The same
>problem has happened to Cy3-filter setting, too, although less
>frequently. I wonder if anyone has a idea about what they are and how
>to prevent them from showing up.
>
>We fix brains in 4%PFA/PB overnight, wash and cryoprotect in sucrose/
>PB, and freeze in OCT in plastic molds on petri dish on liquid
>nitrogen. We cut 20um cryosections, dry them for an hour or so, and
>do regular antibody staining, do DAPI staining, dehydrate, and mount
>in DPX.
>
>Thank you for your help.
>
>Yasushi Nakagawa
>

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