Rabbit monoclonal anti zo 1

Immunostaining was carried out to confirm cell-specific and tissue-specific markers. For this purpose, the cultured cells or cryostat sections were washed with PBS and fixed with 4% paraformaldehyde for 15 min and then washed twice with PBS. Then, they were incubated in PBS-3% bovine serum albumin (BSA)-0.3% Triton X-100 at room temperature for 1 h followed by incubation with primary antibodies (rabbit polyclonal anti-OCT4, 1:400, CST; mouse polyclonal anti-Sox2, 1:200, Abcam; rabbit monoclonal anti-ZO-1, 1:400, Abcam; mouse polyclonal anti-Mitf, 1:400, Santa Cruz; rabbit anti-human nuclear antigen, Abcam; mouse anti-CD68, Abcam; rabbit polyclonal anti-Brn3b, 1:200, Proteintech; rabbit polyclonal anti-RPE65, Abcam) overnight at 4 °C. Following this, the cells or cryostat sections were incubated with goat anti-mouse IgG H&L Alexa Fluor®488/goat anti-rabbit IgG H&L Alexa Fluor®555 (1:500, Abcam) for 2 h at room temperature. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Images were captured with fluorescence microscopy (Observer 7, Zeiss).

Proteins of mouse retina tissue were extracted with a protein extraction solution assay kit (RAPI, Sigma). Nanodrop (Thermo) was used to measure protein concentration. Equal amounts of proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel and 5% stacking gel and transferred onto polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked with skim milk (BD) in Tris-buffered saline (TBS) for 1 h at room temperature. Following this, they were incubated with primary antibodies at 4 °C overnight (rabbit monoclonal anti-ZO-1, 1:500, Abcam; mouse monoclonal anti-RPE65, 1:500 Abcam; mouse polyclonal anti-Nestin, 1:500 Santa Cruz; rabbit monoclonal anti-α-SMA, 1:1000, Abcam; mouse polyclonal anti-CD68, 1:1000, Proteintech; rabbit anti-Bax, 1:1000, Abcam; rabbit monoclonal anti-GAPDH, 1:1000, Abcam). This was followed by PVDF membranes being washed with TBS-T (Tween 1:1000 dilution in TBS buffer) three times followed by incubation with secondary antibodies (HRP-goat anti-rabbit IgG, 1:2000, Bioss; HRP-goat anti-mouse IgG, 1:2000, Bioss) for 1 h at room temperature. After incubation, PVDF membranes were washed three times with PBS. Protein signals were detected with SuperSignal™ West Femto Maximum sensitivity substrate (Thermo Fisher Scientific) and imaged using the chemiluminescence system (Bio-Rad).