Re: [IHC on frozen sections of brain]

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From:"Barry, Lilith" <> (by way of histonet)
To:histonet <>
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Is the tissue  fixed and cryoprotected or it is fresh-frozen? If it is fixed
then thick sections can be collected in buffer and processed free- floating.
I use tissue culture well plates. Make sure  pemeabelize well before
incubation  (I use 0.1% TX  in preblocking solution) and use the same
concentration of antibody as I would use for thin sections. Put small amount
in the wells and incubate overnight on some sort of rotating, shaking  table
in the fridge overnight. This way the antibody penetrates from both sides of
the tissue.
If the tissue is not fixed, with such thick tissue your morphology might not
look good. You can still  process the sections on the slides, use the same
amount of antibody. Make sure to permeabelize and incubate  in the fridge at
least overnight.
If you have other questions, please don't hesitate to contact.
Lilith Ohannessian-Barry
National Research Council
Institute of Biological Sciences

From: Karen S Pawlowski
To: Barry, Lilith
Subject: Re: [IHC on frozen sections of brain]
Date: Friday, December 18, 1998 10:26AM

Your comment caught my eye because I have a friend who is trying to learn
IHC on frozen sections and is having problems with the cryostat.  It has
been fixed (the compressor, etc.) twice in the last 6 months and is out
again.  She is a student and she is affraid she will be in the middle of
her experiment when it will go out again....So, she wants to try using
the sliding microtome and cutting 50-80 micron sections instead of 10
micron sections.  The question she had was will she be able to use the
same amount of antibody on 50 micron versus 10 micron sections? (She is
using a PAP pen and incupating with puddles of solution on the slides.)

I have only done crystat-cut frozens this way and

On Thu, 17 Dec 1998, Barry, Lilith wrote:

> I use Vector ABC kit routinely on perfused brains and it works very well.
> The few times I have tried to use it on fresh-frozen sections, the
> background was very high. Does anybody know why?
>  Lilith

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