Immunohistochemical detection of proliferative cells

The cell proliferation represents one of the most relevant cellular functions. There are many approaches to assess the proliferative activity in tissues. Immunohistochemical methods offer a high sensitivity and specificity. They use monoclonal antibodies raised against specific antigens associated with the cell proliferation. Anti-BrdU immunohistochemistry detects bromodeoxyuridine (BrdU) which as an exogenous proliferation marker has been incorporated into the newly synthesized DNA of dividing cells. The best known endogenous proliferation markers are PCNA and Ki-67 that may be detected with specific commercially available antibodies. Here we describe our experience with an immunohistochemical detection of these proliferation markers. Anti-bromodeoxyuridine (BrdU) antibody binds BrdU that was exogenously introduced into the synthesizing DNA during the S-phase of the cell cycle. The precise timing and dosage of BrdU enable tissue kinetics studies. A long-time administration of BrdU may be used for labelling tissues with a low proliferative activity. BrdU incorporated into the cell nucleus is still a very stable antigen which gives a strong and reliable signal regardless the kind of fixation and further tissue processing. The proliferating cell nuclear antigen (PCNA) as an auxillary protein for DNA polymerase gamma is an evolutionarily conserved molecule that may be detected in human and animal frozen or paraffin-embedded tissues. In spite of the fact that anti-PCNA immunohistochemistry may weakly stain non-proliferating cells, PCNA is the most frequently detected proliferation marker. The staining intensity may be influenced by the kind of fixation and the length of microwave pretreatment. Ki-67 is another endogenous antigen detectable in proliferating cells. Its detection in paraffin-embedded sections requires also exposition to microwaves. Both Ki-67 and PCNA may be revealed in archival specimens that means these techniques do not require any prelabelling. Ki-67 labelling index (LI) correlates better with BrdU LI than PCNA LI as the Ki-67 antigen has a shorter half-life than PCNA.