Histological and Immunofluorescence Analysis of Testicular Tissue

Testicular tissues were fixed in Bouin’s solution for histological analysis via hematoxylin and eosin staining. Testicular tissues were fixed in 4% paraformaldehyde (PFA) for immunofluorescence staining of cross-sections or whole mount seminiferous tubules as described previously [50 (link)]. After antigen retrieval in 10 mM sodium citrate (pH 6.0), the slides were washed and then blocked for 1 h in 10% donkey serum (Solarbio, SL050). The sections were incubated with primary antibody at 4 °C overnight and then incubated with the secondary antibody for 2 h. Specimens were mounted for observation under a Nikon fluorescence microscope (Nikon, ECLIPSE E200, Japan) equipped with a CCD camera (Tusen, China).