Goat serum

TCs were seeded on coverslip following washing with Medium 199 to remove unattached cells after overnight incubation. For the identification of TCs and GCs, a CYP17A1 antibody, a marker enzyme specifically expressed in TC, was used to verify the purity and identify the cells. Icy acetone was added and a coverslip was placed at −20°C for 10 minutes. After washing and permeabilization with PBS containing 0.5% Triton X-100, the cells were blocked with goat serum (Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) for 1 h in a humidified chamber at 37°C. Subsequently, slides were incubated with CYP17A1 antibody (1 : 200, Santa Cruz, CA) or FSHR antibody (1 : 200, Bioworld Technology, Inc., Minnesota) overnight at 4°C. Slides were incubated with secondary fluorescent FITC (1 : 100, Boster Biotechnology Co. Ltd., Wuhan, China) for 1 h in a humidified chamber at 37°C. After washing and nuclear staining with Hoechst for 10 seconds, the staining was stopped. The slides were seal-capped with Debico and observed under a fluorescence microscope.

The integrity of the BTB assessment was evaluated using a biotin tracer as described previously [19 (link)]. In short, the mice were anaesthetized, and the testes were exposed before sacrifice. The gaps below the testicular tunica albuginea were injected with 50 μl of EZ-Link Sulfo-NHS-LC-Biotin (10 mg/ml, freshly dissolved in physiological saline containing 1 mM CaCl₂; Pierce Biotechnology Inc., IL, USA). After 30 min, the animals were euthanized, and their testes were frozen in liquid nitrogen in preparation for cryosectioning (10 μm). The sections were fixed in 4% paraformaldehyde (PFA) for 20 min, washed with phosphate-buffered saline with 0.1% Tween20 (PBST) three times, blocked in 0.01 M phosphate buffer solution (PBS) containing 15% goat serum (Zhongshan Jinqiao Biotechnology Co. Ltd., China) and 1% bovine serum albumin (BSA, wt/vol) for 1 h, incubated with Alexa Fluor® 568-conjugated streptavidin (Life Technologies Corp.-Invitrogen, CA, USA) and 4′-6-diamidino-2-phenylindole (DAPI, blue) (Invitrogen, UK) for 2 h at room temperature, and washed again with PBST five times (5 min per time). After mounting with antifade mounting medium P0128 (Beyotime Biotechnology, China), the sections were analyzed by fluorescence microscopy (Axio Imager A2, Carl Zeiss, Germany).

First, the spleen paraffin sections were deparaffinized in xylene and graded ethanol. To block endogenous peroxidase activity, the sections were incubated with 10% hydrogen peroxide. For antigen retrieval, the sections were heated in 2% EDTA solution for 15 min, and allowed to cool for 2 h at room temperature. After washing with PBS, the slides were blocked with goat serum (Zhongshanjinqiao Biotechnology Co., Ltd., China) for 15 min, and then incubated with the diluted antibodies at 4℃ overnight. On the next day, the sections were incubated with corresponding secondary antibodies at room temperature for 15 min. The sections were then labeled with horseradish peroxidase, and the signals were detected using the DAB Peroxidase Substrate Kit (Solarbio). After staining with hematoxylin (Beyotime) for 30 s, the slides were dehydrated in graded ethanol and xylene. Finally, the sections were sealed with coverslips by resinene (Solarbio). The following primary antibodies were used for the assay: anti-GS (Affinity), anti-mTOR (Affinity), anti-Cyclin D1 (Affinity), anti-CDK4 (Affinity), anti-CDK6 (Affinity).