Light microscopy

The frozen tissue section slides were fixed in acetone for 15 minutes and subsequently washed by PBS. Endogenous peroxidases and non-specific binding were blocked by incubating with 3% hydrogen peroxide for 10 minutes and with 3% BSA, respectively. Slides were incubated with primary antibodies for 30 minutes at room temperature, followed by a 30-minute incubation with horseradish peroxidase-conjugated goat anti human IgG antibodies (Thermofisher). Antibody binding was visualized by incubating with the peroxidase substrate 3,3’-diaminobenzidine (DAB, GeneTech) for 5 minutes. After counterstaining with hematoxylin (Sangon Biotechnology), tissue sections were analyzed using light microscopy (Nikon).

We prepared the transfected cells into cell culture dishes at a density of 300 cells/well. Cells in complete culture medium were cultured in incubators for 12 days. The medium was refreshed every two days to induce colony formation. At last, cell debris was removed by PBS washing, and cell colonies (over 50 cells) after methanol fixation and 0.1% crystal violet (Sangon Biotech, Shanghai, China) staining were observed by light microscopy (Nikon, Japan).