[Histonet] Antibody penetration problem

[Amos Brooks]

Hi Gary,
   I am certainly not an expert on doing this, but I have done it and it
worked fairly well. Why not do this as a free floating section. Take the
section and place it in a glass beaker to with xylene. Deparaffinize and
rehydrate as you would a slide, but not on a slide ... in a beaker. Remember
to agitate the section gently. Once you are out of xylene you might want to
switch to a tissue culture flask (one of those flat rectangular ones with a
cap).
   For antigen retrieval (always avoid the microwave) put the container in a
60 deg oven overnight (cap on of course). That should do the trick. You can
always tweak the procedure later to improve results. Once you get to the
antibody and detection you can use a capped tube on it's side if you keep it
on a gentle shaker table. Once you have the tissue out of chromagen put it
in a dish and float it onto a slide.
   Incidentally weather you do this all on a slide or in a jar to mount
later you need to make sure there is sufficient surfactant in the buffer
rinses. This really aids the penetration by removing the surface tension.
Also make sure your pH is correct thru the whole procedure. Failing this
will affect the antibody antigen affinity (or is it avidity ... I forget).

Best of luck,
(and let us know how it goes)
Amos Brooks


Message: 12
Date: Wed, 11 Feb 2009 15:19:09 -0500
From: Gay Rudow <grudow1 <@t> jhmi.edu>
Subject: [Histonet] Antibody penetration problem
To: "'histonet <@t> lists.utsouthwestern.edu'"
       <histonet <@t> lists.utsouthwestern.edu>
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I hate to change the topic, but I have a problem with penetration of an
antibody. I am using 50 ěM paraffin sections and doing an antigen retrieval
step of microwaving the sections in water for 5 min. I am staining by hand
using the Vector ABC Elite mouse kit. The antibody is SMI 311 which I am
using as a neuronal marker. Right now, my antibody penetration is only 15
ěM. Does anyone have any suggestions to help me with this? Thanks!

Gay Rudow