Anti itga2

Total cell extracts were prepared in radioimmunoprecipitation assay buffer (20 mM Tris-HCL, pH 8.0, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), containing a protease inhibitor cocktail (Sigma-Aldrich, P2714), and incubated at 4°C for 1 h. Western blotting was performed as previously described (Perez-Garcia et al., 2021 (link)). In brief, protein lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore). Membranes were blocked with 5% milk powder and incubated with primary antibody overnight at 4°C, followed by horseradish peroxidise (HRP)-conjugated secondary antibodies. Blots were probed with the following antibodies: anti-ITGA2 (1:1000, Abcam, ab181548), anti-JAG2 (1:1000, Cell Signaling Technology, 2210), anti-Cleaved NOTCH1 (1:1000, Cell Signaling Technology, 4147), anti-NOTCH1 (1:1000, Cell Signaling Technology, 3608), anti-TP63 (1:1000, Abcam, ab124762), anti-IRF7 (1:1000, Cell Signaling Technology, 4920) and anti-β actin (1:5000, Abcam, ab6276). Horseradish peroxidase-conjugated secondary antibodies (used at a dilution of 1:3000) were sourced from Bio-Rad. Detection was carried out with enhanced chemiluminescence reaction (GE Healthcare, RPN2209) using standard X-ray films.

Formalin-fixed, paraffin-embedded tissue samples were sectioned with a standard microtome at 3- to 5 µm thickness. After deparaffinization and rehydration, heat-induced (98°C) antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at a sub-boiling temperature for 10 mins. The slides were incubated with hydrogen peroxide 3% (v/v) for 10 min, washed and blocked with 5% FBS in TBST for 1 hr at room temperature. Next, slides were incubated with primary antibodies anti-COL1A1 (1:100) (Sigma-Aldrich) and anti-ITGA2 (1:500) (Abcam) at 4°C overnight. Primary antibodies were detected using a SignalStain Boost IHC anti-rabbit HRP Reagent (Cell signaling Technology). The signal was visualized using a diaminobenzidine substrate kit (DAB, Thermo Fisher Scientific) according to the manufacturer's instructions and nuclei were counterstained with hematoxylin. Immunostaining was scored by the weighted average score (intensity: 0–3, coloring: 0–100% of ITGA2 expression) by two trained scientists independently and discrepancies were resolved by consensus.