Anti occludin antibody

Calcitriol, ethanol, and 4′,6-diamidino-2-phenylindole dihydrochloride n-hydrate (DAPI) were obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Dfb ointment (Biostir-AD) was purchased from Biostir Inc. (Osaka, Japan).
Primary antibodies against filaggrin (1:1000 dilution), involucrin (1:1000 dilution), and loricrin (1:1000 dilution) were purchased from BioLegend (San Diego, CA, USA). Anti-claudin-1 antibody (1:20,000 dilution) was obtained from Spring Bioscience (Pleasanton, CA, USA), anti-occludin antibody (1:1000 dilution) was obtained from Cell Signaling Technology (Beverly, MA, USA), and anti-ZO-1 antibody (1:4000 dilution) was purchased from Proteintech (Rosemont, IL, USA). The secondary antibodies conjugated to Alexa Fluor dye were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and diluted 1:1000.

Immunofluorescent analyses of tight junctions were performed on Bouin’s fixed paraffin-embedded testicular sections (5 µM). Heat treatment in a sodium citrate-based antigen retrieval system was used, followed by incubation in 3% hydrogen peroxide for 15 min to quench hydrogen peroxidase activity. Nonspecific binding was blocked using normal horse serum diluted 1:4 in PBS with 5% BSA (Bovine Sum Albumin). Testis sections were then incubated with anti-ZO-1 antibody (Cat # 617300; Invitrogen) diluted 1:100 blocking solution overnight at 4°C, followed by a 1-h room temperature incubation in Alexa Fluor 488-conjugated anti-rabbit antibody (1:500; Life Technologies). Anti-occludin antibody (Cat No. E6134R; Cell Signaling) diluted 1:200 in blocking solution overnight at 4°C, followed by incubation for 1 h at room temperature with Alexa Fluor 488-conjugated anti-mouse antibody (1:500; Life Technologies). Anti-caspase 3 antibody (Cat No. 9664L; Cell Signaling) diluted 1:100 in blocking solution overnight at 4°C, followed by a 1-h room temperature incubation in Alexa Fluor 488-conjugated anti-rabbit antibody (1:500; Life Technologies). Sections were mounted with VectaShield Mounting medium (Vector Labs). All sections were imaged as described above.