Mouse e cadherin antibody

Cells were harvested in RIPA Lysis Buffer (strong) (CWbio, Taizhou, China; CW2333S) containing protease inhibitors (Roche, 4693124001). A total of 40 µg proteins were separated by 4%–20% gradient SDS-PAGE gels (GenScript, M42015C) and transferred to PVDF membranes (Millipore, Billerica, MA, USA; IPVH00010). The membranes were blocked at room temperature with 1% BSA for 1 h and incubated overnight at 4 °C with primary antibodies: rabbit Collagen I antibody (Cell Signaling Technology, 84336), mouse E-cadherin antibody (Abcam, ab1416), mouse α-SMA antibody (Sigma-Aldrich, A5228) and mouse β-actin antibody (Sigma-Aldrich, A1978). Then, the membranes were washed with TBST for 3 times and incubated for 1 h with a secondary antibody: anti-mouse IgG antibody (HRD) (Sigma-Aldrich, A9044) and anti-rabbit IgG antibody (HRD) (Sigma-Aldrich, A0545) at room temperature. Images were obtained using the ChemiDoc XRS + imaging system (Bio-Rad) and quantified using the Quantity One software.

Paraffin embedded tissue sections were de-waxed with a double 5-min washes in Xylene followed by rehydration with 5-min each 100% ethanol, 95% ethanol, 70% ethanol, and distilled water washes. Slides were microwaved at high temperature in sodium citrate buffer solution (10 mM citric acid and 0.05% Tween-20, pH 6.0) for 30 min and then allowed to cool for antigen retrieval. After PBS washes, samples were incubated with PBS-T (PBS with 0.01% Tween-20) containing 10% KPL blocker (milk diluent/blocking solution concentrate, Seracare) to block nonspecific binding sites followed by incubation with mouse-E-cadherin antibody (Abcam, ab231303) or rabbit anti-Iba1 antibody (NovaChem, 019-19741) (both 1:500 dilution in 5% KPL buffer) in a humidified chamber at 4 °C overnight. Samples were then rinsed with PBS-T, incubated with anti-rabbit AF647 (#A-21244) or AF488 (#A-11008) secondary antibodies for 1 hr at room temperature (1:500 5% KPL buffer, ThermoFisher Scientific) and counter stained with DAPI (Sigma-Aldrich, 1:1000) for 15 min. The slides were mounted with cover slips and analysed under confocal laser scanning microscope (Olympus FV3000). Confocal images were analysed for relative quantification of fluorescent markers by ImageJ software (version 1.53).