Optimizing immunohistochemical staining for the HGF receptor c-MET in formalin-fixed paraffin-embedded archival human tissue

Frozen tissue immunohistochemistry is a useful technique employed in diagnostic and research investigations and is often used despite a loss of morphological detail. Formalin fixation yields much better tissue preservation, but may mask the antigen sites. We have optimized a technique that allows us to study the distribution of the HGF receptor, c-MET, in formalin fixed, paraffin embedded tissue using commercially available polyclonal antisera in which we unmasked antigens sites with protease digestion. The technique we use is detailed below.Sections are incubated in the following:1)4 μm paraffin sections are deparaffinized and hydrated2)0.03% protease (Type XXIV: Sigma) for 2 min at RT3)0.5% H2O2 in methanol for 30 min at RT4)Protein Blocking Agent (Lipshaw/Immunon) for 1 hr at RT5)1° antibody ( Santa Cruz Biotech, Santa Cruz, CA) for 2 hr at RTa.)c-MET (c-12) rabbit polyclonal IgG, 1:200 dilution in PBSb.)h-MET (c-28) rabbit polyclonal IgG, 1:100 dilution in PBSc.)m-MET (c-21) rabbit polyclonal IgG, 1:100 dilution in PBS