Stem cell factor (scf)

For immunocytochemistry staining, freshly isolated and cultured human Sertoli cells were fixed with 4% paraformaldehyde (PFA) for 30 min, washed three times with cold PBS (Medicago, Uppsala, Sweden), and permeabilized with 0.4% Triton X-100 (Sigma) for 5 min. After extensive wash with PBS, the cells were blocked in 5% bovine serum albumin (BSA) (Sigma) for an hour at room temperature. The cells were then incubated with primary antibodies overnight at 4°C, including GATA4 (1:200; Santa Cruz, Dallas, TX, USA), WT1 (1:200; Santa Cruz, Dallas, TX, USA), SOX9 (1:500; Millipore, Bedford, MA, USA), GDNF (1:300; Santa Cruz, Dallas, TX, USA), SCF (1:300; Santa Cruz, Dallas, TX, USA), VIM (1:100; Cell Signaling Technology [CST], Danvers, MA, USA), OCLN (1:200; Abcam, Cambridge, UK), α-SMA (1:200; Abcam, Cambridge, UK), CYP11A1 (1:200; Abcam, Cambridge, UK), VASA (1:100; Santa Cruz, Dallas, TX, USA), and ki-67 (1:200; Santa Cruz, Dallas, TX, USA). After extensive washes with PBS, the cells were incubated with the secondary antibody, namely immunoglobulin G (IgG) conjugated with fluorescein isothiocyanate (FITC) (Sigma) or rhodamine-conjugated IgG (Sigma), at a 1:200 dilution for 1 hr at room temperature. DAPI was used to label the nuclei. Replacement of primary antibodies with PBS was used as a negative control, and images were captured with a Nikon microscope.