Uchl1

The immortalized human male germline stem cells and human primary SSCs were lysed with RIPA buffer (Santa Cruz) for 30 min on ice. Cell lysates were cleared by centrifugation at 12,000 g, and the concentrations of total proteins were measured by BCA kit (Dingguo Company, China). Twenty micrograms of cell lysates from each sample were resolved by SDS-PAGE (Bio-Rad Laboratories, Richmond, CA), and Western blots were performed according to the protocol as described previously1 (link). The chosen antibody included SV40 (Santa Cruz), PCNA (Santa Cruz), RET (Santa Cruz), UCHL1 (AbD Serotec), and ACTB (Proteintech). After extensive washes in PBS, the blots were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad, Hercules, CA).

Human Sertoli cells were isolated from obstructive azoospermia patients with normal spermatogenesis using a two-step enzymatic digestion and followed by differential plating. Immunocytochemistry was performed to determine the identity of human SSC line, primary human Sertoli cells, and mouse liver mesenchymal cells pursuant to the procedure described previously [37 ]. Briefly, cells were fixed in 4% paraformaldehyde (PFA) for 30 min and followed by permeabilization with 0.4% Triton X-100 for 15 min and blocking in 1% bovine serum albumin (BSA, Sigma-Aldrich) for 1 h. The primary antibodies were as follows: UCHL1 (AbD Serotec), GFRA1 (Santa Cruz), GPR125 (Abcam), PLZF (Santa Cruz), VIMENTIN (Abcam), and VWF (Santa Cruz). After incubation overnight at 4°C, rhodamine-conjugated or FITC-conjugated IgG was used as secondary antibodies. The dilutions, resources, and companies of primary and secondary antibodies for immunocytochemistry were shown in Table 2. Double immunostaining was also performed to verify the identity of human SSC line antibodies against GFRA1 and UCHL1 as well as GPR125 and UCHL1. Replacement of primary antibodies with isotype IgG or PBS served as a negative control. The nuclei of cells were stained with DAPI (4′-6-diamidino-2-phenylindole) and the epifluorescence were examined under fluorescence microscope (Nikon Eclipse Ti-S, Nikon Corporation, Tokyo, Japan).

For immunocytochemical staining, the freshly isolated human spermatogonia, pachytene spermatocytes and round spermatids were fixed with 4% paraformaldehyde (PFA) for 30 min, washed three times with cold PBS and permeabilized in 0.4% triton-X 100 (Sigma-Aldrich) for 5 min. After washing with PBS, the cells were blocked in 1% BSA for 30 min and followed by incubation with primary antibodies, including GPR125 (Abcam), GFRA1 (Abcam), UCHL1 (AbD Serotec), MAGEA4 (melanoma antigen family A, 4, a kind gift from Professor Giulio C. Spagnoli, University Hospital of Basel, Switzerland), SCP3 (Abcam), Acrosin (Santa Cruz), or Protamine 2 (Santa Cruz) at a dilution with 1:200 overnight at 4°C. After extensive washes with PBS for 30 min, the cells were incubated with the secondary antibody IgG (Sigma) conjugated with fluorescein isothiocyanate (FITC) or rhodamine at a 1:200 dilution for 1 hour at room temperature. DAPI (4,6-diamidino-2-phenylindole) was used to label the nuclei, and the cells were observed for epifluorescence under a fluorescence microscope (Leica). For the detection of MAGEA4, peroxidase-conjugated goat anti-mouse IgG (Envision detection kit, DAKO) was used as the secondary antibody and examined under a light microscope.

Immunofluorescence staining was performed to determine the identity of cultured hSSCs and the differentiated hSSCs by the induction system pursuant to the procedure as described previously [27 (link)]. Briefly, cells on coverslips or plates were fixed in 4% paraformaldehyde for 20 min, permeabilized with or without 0.01% Triton X-100 (Sigma) for 10 min, and blocked in 4% normal donkey or goat serum at room temperature. Concomitantly, cells were incubated with primary antibodies at a dilution of 1:200–1000, respectively, overnight at 4 °C. The primary antibodies used in this study were as follows: UCHL1 (AbDSerotec), GFRA1, EN-1, OTX2 (Santa Cruz), GPR125, Tuj-1, PLZF, FoxA2 Lmx1a, Lmx1b (Abcam), DAT (Millipore), Curr1 (Millipore), PitX3 (GeneTex), and SYN (Chemicon). After three washes in PBS, the cells were incubated with corresponding Alexa Fluor® 488, Alexa Fluor® 594, or FITC-conjugated-IgG secondary antibodies at a 1:200–1:500 dilution for 1 h at RT. DAPI (4″-6-diamidino-2-phenylindole) was used to stain cell nuclei, and the cells were observed under a fluorescence microscope (Olympus BX-53, Japan).