Abstract
DNA replication occurs during S-phase in spermatogonia and preleptotene spermatocytes during spermatogenesis. 5-Bromo-2'-deoxyuridine (BrdU) is incorporated into synthesized DNA and is detectable in the nucleus by immunohistochemistry (IHC). To identify BrdU-labeled spermatogenic cells, the spermatogenic stages must be determined by visualizing acrosomes and detecting cell type-specific marker molecules in the seminiferous tubules. However, the antibody reaction with BrdU routinely requires denaturation of the DNA, which is achieved by pretreating tissue sections with hydrochloric acid; however, this commonly interferes with further histochemical approaches. Therefore, we examined optimal methods for pretreating paraffin sections of the mouse testis to detect incorporated BrdU by an antibody and, at the same time, visualize acrosomes with peanut agglutinin (PNA) or detect several marker molecules with antibodies. We found that the use of heat-induced antigen retrieval (HIAR), which consisted of heating at 95C in 20 mM Tris-HCl buffer (pH 9.0) for 15 min, was superior to the use of 2 N hydrochloric acid for 90 min at room temperature in terms of the quality of subsequent PNA-lectin histochemistry with double IHC for BrdU and an appropriate stage marker protein. With this method, we identified BrdU-labeled spermatogenic cells during mouse spermatogenesis as A1 spermatogonia through to preleptotene spermatocytes.