4 6 diamidino 2 phenylindole (dapi)

In order to determine the location and quantity of the specific antigens in the interspace skin following burn injury, the prepared slices were washed in PBS for 10 min, followed by boiling at 95°C in 0.01 mmol citrate buffer (pH, 6.0) for 10 min for antigen retrieval. Then, the slices was incubated with hydrogen peroxide for 10 min at room temperature, and 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) was applied later as the blocking solution for 20 min at room temperature. Without being washed, the sections were incubated with anti-phosphorylated (p)-Erk (1:200; cat. no. 4370; Cell Signaling Technology, Inc.) or anti-p-Bad (1:200; cat. no. sc-12969-R; Santa Cruz Biotechnology, Inc.) antibodies overnight at 4°C. After being rinsed with PBS, the sections were incubated with FITC-(1:50; cat. no. BA1105; Wuhan Boster Biological Technology, Ltd.) or Cy3-(1:50; cat. no. BA1032; Wuhan Boster Biological Technology, Ltd.) labeled goat anti-rabbit secondary antibodies for 2 h at 37°C in the dark. The sections were rinsed and stained with DAPI (100 ng/ml; Wuhan Boster Biological Technology, Ltd.) for 8 min at room temperature, and then mounted with VECTASHIELD^® mounting medium (Wuhan Boster Biological Technology, Ltd.). All slices were observed and photographed under a fluorescence microscope (magnification, ×200) (DM5500B; Leica Microsystems GmbH).

Cells (1×10⁵/ml) were fixed in 4% paraformaldehyde for 20 min at room temperature and permeabilized using 0.1% Triton X-100 for 20 min. Samples were blocked in goat serum (Wuhan Boster Biological Technology, Ltd.; cat. no. AR009) for 30 min and subsequently incubated with the following primary antibodies: Anti-β-catenin (Wanleibio Co., Ltd.; cat. no. WL0962a; 1:200) and anti-mono-ADP-ribose binding reagent (EMD Millipore; cat. no. MABE1076; 1:200) at 4°C overnight. The following day, cells were washed in PBS and incubated with Cy3-conjugated secondary antibody (ProteinTech Group, Inc.; cat. no. SA00009-2; 1:200) in the dark for 1 h at room temperature. DAPI (Wuhan Boster Biological Technology, Ltd.) was used for nuclear staining (5 min at room temperature). The images were captured using ZOETM Fluorescent Cell Imager (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ software (version 1.48; National Institutes of Health) The experiment was repeated three times.