[Histonet] Re: Problem with liver fixation

[Andrea Marion]

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