Immunohistochemical detection of c-myc oncoprotein in paraffin embedded tissues

In almost all studies using paraffin embedded tissue, c-myc protein has been found in the cytoplasm of cells. Since the protein is normally localized in the nucleus it is difficult to determine which histochemical observations are real and which are artefactual. The study designed here evaluated several different methods of fixation prior to paraffin embedding in an attempt to identify which would prevent the diffuse of the protein out of the nucleus. Using various fixation procedures (formalin, paraformaldehyde, B-5, Zamboni and AMeX) we found that fixation in cold acetone (-20 degrees C) overnight followed by 2x15 min fixation in acetone at +4 degrees c and at room temperature, cleared in methyl benzoate and xylene (AMeX procedure) gives reproducible nuclear staining when a variety of normal and tumor tissues are treated with an anti c-myc protein antibody. This method was then compared to frozen sections. While there was no cytoplasmic staining in same tissue specimens in both AMeX processed and frozen sections, the tissue architecture was much better preserved in AMeX processed samples. Our data strongly suggest that AMeX fixation, originally developed for T and B lymphocyte antigens, should be used for immunolocalization of c-myc oncoprotein in paraffin embedded tissues.