Stra8

IHC was performed as described in (21 (link)). Briefly, after deparaffinization and rehydration of paraffin sections following standard procedures, heat induced epitope retrieval was carried out in sodium citrate antigen unmasking buffer (pH 6.0) using a decloaking chamber (Biocare Medical, CA, USA) at 110˚C for 20 min. After inactivation of endogenous peroxidases with 0.3% H₂O₂ in absolute methanol and blocking with 10% goat serum in phosphate buffer saline, primary antibody (LKB1, 1:100 dilution, Plzf, 1:100 dilution, Santa Cruz Biotechnology, CA, USA; mTOR and p4EBP1, 1:100, Cell Signaling Technology, MA, USA; Stra8, 1:100 dilution, Abcam, VIC, Australia) was applied to the sections. Following incubation in HRP-tagged secondary goat anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, PA, USA), the color was developed with DAB (3,3’-diaminobenzidine, Sigma-Aldrich) (21 (link)). For immunofluorescence, fluorophore tagged secondary antibodies (Jackson ImmunoResearch Laboratories, PA, USA) were used (26 (link)).

Cells on Matrigel (BD Bioscience) coated cover slides were fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) at 4 °C overnight, and immunofluorescence (IF) assays were performed according to published protocols^{55 (link)}. The following antibodies were used: PLZF (SC-28319, Santa Cruz Biotech, Dallas, TX, USA), STRA8 (ab49602, Abcam, Cambridge, MA, USA), SYCP3 (ab15093, Abcam), Alexa Fluor 488- or TRITC-conjugated anti-mouse and anti-rabbit secondary antibodies (115-545-146, 115-025-146; 111-545-144, and 111-025-144, Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with 0.5 µg/mL DAPI before being visualized using an Olympus microscope BX53. Images were processed with the Image-J software. For histology studies, testes were fixed in Bouin’s fixative at 4 °C overnight for staining with hematoxylin and eosin, as previously described^{56 (link)}. For immunohistofluorescence (IHF), testes were fixed with 4% paraformaldehyde in PBS at 4 °C overnight and embedded in paraffin. Testis sections were stained with an ACR antibody (HPA048687, Atlas Antibodies, Sweden), followed by staining with an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch) and Rhodamine-labeled Peanut Agglutinin/PNA (RL-1072, Vector Laboratories, USA). Images were collected using a fluorescent microscope (Leica, DM400BLED368424).

The mouse testes of the three groups after hAMSC treatment and the Sertoli cells of the three groups were harvested for western blotting as described previously [18 (link)]. Antibodies for BCL2, SURVIVIN, CASPASE9, CASPASE3, and GAPDH, which were obtained from Abcam (USA), were used for Sertoli cells. Antibodies for BCL2, SURVIVIN, CASPASE9, CASPASE3, Dazl, Ddx4, Miwi, Scp3, Cyclin A1, Stra8, GAPDH, and β-Actin were obtained from Abcam (USA) and were used for the testes.