Ivana:
Have you considered performing any pretreatment procedures, like enzymatic  
digestion or heat-retrieval?
Joe
 
------------------------------

Message: 9
Date: Tue, 11 Mar 2008  02:43:05 -0700 (PDT)
From: Ivana Nikic  
Subject: [Histonet] Antibody penetration  issues
To: histonet@lists.utsouthwestern.edu

Hello everybody,
I am  having some difficulties with my stainings in the mouse spinal cord. I 
am using  longitudinal, both vibratome and cryo-, 4%PFA-fixed sections. The 
problem is  that, with protocols I have tried so far, using longitudinal 
sections results in  a non-uniform staining pattern. It works pretty fine in the grey 
matter, but the  penetration in the white matter axons is pretty low. I am 
trying to work this  out by using tissue from transgenic mouse lines with 
fluorescently labelled  neurons and neuronal mitochondria and anti-GFP antibody. I 
have tried X-100  Triton (0.1-1%) permeabilization and several fixatives - 
methanol,  methanol+acetone, ethanol-acetic acid. It helps with the axons, but it 
is still  hard to get the antibody into mitochondria. 
Any feedback would be  greatly appreciated !
Thanks a lot in  advance, 
Ivana




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