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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
