Fluorescence secondary antibody

Cells were fixed with 4% paraformaldehyde (Sigma) for 15 min and permeated with 0.2% Triton X‐100 for 5 min. After washed with PBS buffer three times for 5 min, cells were blocked with fetal bovine serum for 30 min, then incubated with the primary antibody at 4°C overnight [E‐cadherin (1:100, CST, #3195); MAP2 (1:40, Sangon Biotech, AF2215)]. Cells were washed with PBS buffer three times for 5 min and incubated with fluorescence secondary antibody (1:1000, sigma) for 30 min. After washing with PBS buffer, the cells were stained with DAPI (Thermo Fisher Scientific) for 10 min and washed with PBS buffer three times for 5 min before photographed with Inverted Fluorescence Microscope.

Cells and skin tissue samples were fixed in 4% paraformaldehyde overnight. Skin tissue samples underwent dehydration with 30% saccharose and were embedded in optimal cutting temperature compound (OCT) (Leica, Germany) and cut into 10-μm-thick sections. The cells and sections underwent permeabilization with 0.05% Triton X-100 (Sigma-Aldrich, USA) for 10 min at room temperature, blockage with 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA) at 37 °C for 30 min, and incubation with the primary antibody overnight at 4 °C. Next, the sections were incubated with fluorescence secondary antibody (Sigma-Aldrich, USA) at room temperature for 1 h. Lastly, the nuclei were counterstained by Hoechst 33342 (Sigma-Aldrich, USA) for 10 min at room temperature. Photographs were taken by a confocal microscope and evaluated by ImageJ software. The primary antibodies involved in this study include F4/80 (1:200, ab6640, monoclonal), Cytokeratin 14 (1:400, ab181595, monoclonal), CD206 (1:200, ab195191, monoclonal), and Ki67 (1:150, ab16667, monoclonal) (all from Abcam, UK).