Figure 3.

Fluorescence images of mouse seminiferous tubules showing the effects of different pretreatments on double immunostaining of BrdU (red) and various marker proteins (green). Nuclei were counterstained with Hoechst 33258 (blue). Paraffin sections of the testis were pretreated with HCl (A–C, G–I, M–O) or heat-induced antigen retrieval (HIAR; D–F, J–L, P–R), and then immunostained with the mouse monoclonal anti-BrdU antibody together with the rabbit polyclonal anti-PLZF antibody (A–F), rabbit polyclonal anti-CADM1 antibody (G–L), or goat polyclonal anti-GATA4 antibody (M–R). Pictures in different combinations of wavelengths are shown. One cell was simultaneously immunopositive for BrdU (red arrowhead, D) and PLZF (green arrowhead, E) when the photographs were merged (yellow arrowhead, F). The immunoreactivity of BrdU (red; G, J) and CADM1 (green; H, K) mostly overlapped (yellow; I, L). The immunoreactivity of BrdU (red; M, P) and GATA4 (green; N, Q) never overlapped (O, R). Both the immunoreactivity of BrdU (A, D, G, J, M, P) and marker proteins (B, E, H, K, N, Q) were much stronger and clearer in HIAR-pretreated sections (D-F, J-L, P-R) than in HCl-pretreated sections (A-C, G-I, M-O). Scale, 25 μm.