Af332

Adult male C57BL/6 mice were sacrificed by cervical dislocation, and testes were dissociated, cut transversely, and fixed in Bouin for 8 h at 4°C. The tissue was then dehydrated, embedded in paraffin, and cut into 5-μm sections following deparaffinization. Testis sections were treated with 100 mmol l^-1 glycine at room temperature for 15 min and then 3% H₂O₂ for 10 min. The sections were then blocked with normal goat or rabbit serum (SP KIT-B2 or SP KIT-B5; Maixin Biotech, Fuzhou, China) for 2 h at room temperature. Next, the sections were incubated with primary antibodies, including rabbit anti-PLZF (1:200 dilution; SC-22839, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-c-KIT (1:50 dilution; AF332, R&D Systems, Minneapolis, MN, USA), and goat anti-GFRA1 (1:50 dilution; AF714, R&D Systems) for 3 days at 4°C. Immunolabeling was detected by using biotinylated-linked secondary antibodies (SP KIT-C8 or SP KIT-C10; Maixin Biotech) and streptavidin-conjugated horseradish peroxidase (SP KIT-D2, Maixin Biotech). Sections were visualized using 3, 3’-diaminobenzidene hydrochloride (DAB; ZLI-9018, ZSGB-BIO, Beijing, China) and counterstained using Harris hematoxylin (BA4097, BASO, Zhuhai, China). All IHC sections were examined under a Sunny RX50 microscope (Sunny Optical Tech, Ningbo, China).