Vectastain abc and dab peroxidase substrate kits

To achieve better histology of testis seminiferous tubules, testes were fixed in Bouin’s solution and subjected to H&E staining. For IHC staining, samples were fixed in 3.7% phosphate-buffered saline (PBS) buffered formaldehyde. After fixation, the tissues were dehydrated through an ethanol gradient and embedded in paraffin. The samples were sectioned at a thickness of 5 μm. For H&E staining or IHC, sections were deparaffinized and rehydrated. For IHC, sections were treated with 3% hydrogen peroxide for 10 min at room temperature and with 20 mM sodium citrate for 15 min at 95 °C, followed by overnight cooling. Primary antibodies were applied at suitable dilutions (Supplementary Data 1) at room temperature for 1 h, followed by incubation with biotinylated secondary antibodies for 30 min. Sections were then stained using Vectastain ABC and DAB peroxidase substrate kits (Vector Laboratories, Burlingame, CA). The antibodies used in this study are listed in Supplementary Data 1.

Ovary samples embedded in paraffin were sectioned (5 μm thick) for subsequent hematoxylin and eosin (HE) staining and immunohistochemistry (IHC). For IHC, sections were deparaffinized and rehydrated. Primary antibodies were applied at suitable dilutions at room temperature for 1 h, and samples were then incubated with biotinylated secondary antibodies for 30 min. The sections were then stained with Vectastain ABC and DAB peroxidase substrate kits (Vector Laboratories). The images were taken with a microscope system (Carl Zeiss). The diameters of oocytes and follicles were analyzed using Image J.