3 3 diaminobenzidine (dab)

Paraffin-embedded tumor tissues were sliced (0.25 µm), dewaxed using xylene and rehydrated using a series of decreasing concentrations of ethanol solutions, and then incubated with anti-Ki-67 antibody (cat. no. ab16667; 1:250; Abcam) at 4°C overnight. Subsequently, tissue sections were incubated with the goat anti-rabbit IgG secondary antibody (cat. no. A0208; 1:50; Beyotime Institute of Biotechnology) at 37°C for 1 h. After counterstaining with hematoxylin at room temperature for 3 min, color development was performed using DAB (Beyotime Institute of Biotechnology).

IHC was used for detecting Gli-1 in human esophagus carcinoma tissues and the adjacent tissues. Tissue slices were made and estimated by 2 pathologists, with consensus. Slices were dried at 68°C for 20 min. Regular de-waxing and gradual ethanol hydration were performed, followed by incubation with 3% H₂O₂ at 37°C for 10 min. Washing with phosphate-buffered solution (PBS) (Solarbio Life Sciences, Inc. Beijing, China) was followed by boiling the slices with citrate buffer solution (0.01 M) (Boster Biological Technology Co., Wuhan, China) at 95°C for 20 min. After cooling to 25°C, we blocked the slices with normal goat serum (Beijing China Ocean Co.) at 37°C for 10 min. Incubation was performed with rabbit anti-Gli-1 polyclonal antibody (1: 500) (ab151796, Abcam, Shanghai, China) at 4°C overnight. After washing with PBS, slices were incubated with goat anti-Rat H&L (1: 200) (Abcam) at 37°C for 30 min. DAB (Beyotime Biotechnology, Shanghai, China) was used for staining at 25°C for 3~30 min until coloring. Hematoxylin-eosin (HE) (Beyotime) was for staining at 25°C for 2 min. Regular dehydration was carried out, and we sealed the slices with neutral resin (Bioway Biotechnology Co., Beijing, China). We observed the results under a microscope (Olympus Corporation, Beijing, China).