RE: GFAP on Frozen sections- problems

From:"Gutierrez, Juan"

What dilution of antibody are you using?  Is it the same as paraffin?  If it
is you might want to start by cutting it in half.  i.e.1:200 to 1:400.  Have
you tried Gill's III hematoxylin?
Hope this help you a bit.

Juan C. Gutierrez, HT(ASCP)
Histology Supervisor
Christus Santa Rosa Healthcare

-----Original Message-----
From: Miller, Janet []
Sent: Thursday, April 17, 2003 3:48 PM
To: Histonet (E-mail)
Subject: GFAP on Frozen sections- problems

Hi, We are having troubles in our lab trying to get GFAP to work on our
frozen sections.  We use GFAP from DAKO, and it works wonderful on paraffin
sections, has anyone had luck with this antibody working with frozen
sections?  If so, what are your incubation times?  
We are using rat brain, perfused with 4% paraformeldehyde solution, and
cryoprotecting cells with a cryoprotectant solution (containing dwater,
sucrose, Ethylene Glycol, polyvinylpyrrolidine (PVP 30), and sodium
phosphate (both mono and dibasic).  We then cut at 30 uM.  Before staining,
the sections are washed for 1 hour, while gently rocking and changing PBS it
is being washed in.  We would like to counterstain with Hematoxylin, but are
finding that the staining time has to be about 1 min, or the background is
too dark.  This time in Hematoxylin does not seem to be adequate to stain
cells though!  The GFAP signal (developed with AEC) is also very week, and
increasing incubation times does not seem to help intensify the signal, nor
does increasing the time in color development (AEC).  
Does anyone have any suggestions, or another antibody that you have had luck
with while doing frozen sections?  Any help is greatly appreciated! 

Thanks in advance-


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