Anti ocln

For immunocytochemical staining, the immortalized human Sertoli cells were fixed with 4% paraformaldehyde (PFA) for 15 min, washed three times with phosphate-buffered saline (PBS) for 5 min each and permeabilized in 0.5% triton-X 100 (Sigma) for 20 min. After washing with PBS, the cells were blocked in 3% serum or BSA for 60 min and followed by incubation with primary antibodies, including anti-SOX9 (Millipore), anti-WT1 (Santa Cruz), anti-OCLN (Abcam), anti-VIM (vimentin, Santa Cruz), anti-SCF (Sigma), anti-BMP4 (Abcam), anti-GDNF (Abcam), anti-3β-HSD (Santa Cruz), anti-VASA(Santa Cruz), and anti-Ki-67 (BD Biosciences), overnight at 4°C. Replacement of primary antibodies with isotype IgGs (Santa Cruz) served as negative controls. After washing three times in PBS for 10 min each, the cells were incubated with the secondary antibody (Sigma) conjugated with rhodamine at a 1:200 dilution for 1 hour at room temperature. DAPI (4, 6-diamidino-2-phenylindole, Sigma) was used to counterstain the nuclei, and images were captured with a fluorescence microscope (Nikon).

The standard protocol of western blot was performed according to our previous reports.^{26 (link),28 (link)} Briefly, the concentration of total protein from segments of small intestine (ileum) was measured with a BCA protein assay kit. 20 µg of protein was fractionated in 12% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% freshly prepared milk-TBST for 2 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: anti-Ocln (1:2000; Abcam, Cambridge, USA), anti-Tjp1 (1:1000; Invitrogen, Carlsbad, CA) and anti-Cldn4 (1:3000; Abcam, Cambridge, USA). The membranes were then washed three times for 10 min in TBST and incubated with secondary antibody conjugated with HRP for 2 h. Finally, the membranes were subjected to a chemiluminescence detection system and exposed to a photographic film. Immunoreactsive bands were quantified using Image J software and bands were normalized with β-actin.