[Histonet] Re: Problem with liver fixation

Andrea Marion amario3 <@t> uic.edu
Mon Sep 6 21:01:50 CDT 2010


Hi Itai,

Liver antigens tend to degrade rapidly, so immediate fixation is
necessary. It sounds like you are getting the tissue into the solution
quickly enough. Fixation occurs more slowly at 4 degrees than room
temperature though - perhaps your antigen is degrading during this
'slower' fixation? Are you using a sufficient volume of fixative compared
to tissue mass?

Another concern is that some antigens require paraformaldehyde fixation
(ie a 4% solution of buffered formaldehyde prepared directly from solid
paraformaldehyde). Solutions of formaldehyde marketed as '37%
formaldehyde' or formalin typically contain ~10% methanol added as a
stabilizer, which can interfere with some antigens. For
immunohistochemistry purposes, you may need to prepare your formaldehyde
solution directly from paraformaldehye. See here for more discussion of
formaldehyde solutions: http://publish.uwo.ca/~jkiernan/formglut.htm

Finally, you list 'IHC protocol', but why are you sure your problem lies
in the tissue fixation/processing and not the staining protocol? You
probably need to do some form of antigen retrieval - what method are you
using? What is your primary antibody dilution? Has your antibody been
validated for use with IHC/IF? Many antibodies simply do not work with
FFPE tissues. There are many other steps that could be causing trouble... 
Here is a good beginner's guide for IHC with FFPE tissues if you need:
http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf

As a side note, your processing steps may be a little long, but I am not
an expert. We use only 30 minutes for each dehydration and clearing step,
and 2 paraffin steps for one hour each with mouse tissue. Good luck!

Andrea Marion

amario3 &at& uic &dot& edu
Graduate Student
University of Illinois at Chicago







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