Re: GFAP on Frozen sections- problems

From:Geoff McAuliffe

Hi Janet:

    I have used DAKO's rabbit anti-cow GFAP for many years on both 
frozen or paraffin sections, great stuff! Here is how I do frozen 
sections of rat or mouse brain:

Fix in phosphate-buffered 4% paraformaldehyde (perfusion if at all 
possible) for 4-8 hours. Fixation is limited due to sensitivity of other 
antigens I will want to detect..
Rinse in buffer 3-4 changes over several hours.
Cryoprotect in 10%, then 20% sucrose in buffer.
Snap freeze and cut at 20 microns on a sliding microtome.
Collect sections in PBS.
Rinse several times in PBS.
My immunostaining proceedure is very ordinary, not worth repeating.
I dilute the DAKO ab 1:60,000 (not a typo) and stain on a shaker table 
(gently agitation) overnight at room temp.
Vector Elite ABC kit as per instructions. With the older (non-Elite) kit 
my dilutions were about 10X more concentrated (about 1:5,000)
Detect with DAB with intensification with nickel+cobalt or DAB followed 
by Vector's "Iintense"
For counterstaining I like either nuclear fast red after DAB 
nickel+cobalt  OR very dilute Toluidine blue (0.025%) for 5-10 minutes 
followed by acetone dehydration (alcohol removes the Tol blue instantly) 
followed by fresh (no residual alcohols) xlyene after the Vector Intense 
(gold cells with blue nuclei, very pretty).


Miller, Janet wrote:

>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-

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

<< Previous Message | Next Message >>