Immunohistochemistry for aldehyde dehydrogenase 1A1 (ALDH1A1)

Tissue sections were obtained from the prepared TMAs. The expression of aldehyde dehydrogenase 1A1 (ALDH1A1) protein was detected using immunohistochemistry. Tissue sections were deparaffinized and rehydrated using xylene and alcohols, and incubated in 3% diluted hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed using an autoclave method, followed by incubation with the primary antibody overnight at 4°C in a high humidity cabinet. The primary rabbit anti-ALDH1A1 polyclonal antibody (clone No. ab52492) (Abcam, Cambridge MA, USA) was used at a dilution of 1: 400 in phosphate-buffered saline (PBS). The secondary horseradish peroxidase (HRP)-conjugated antibody (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) was used according to the manufacturer’s instructions. Negative controls used included replacing the primary antibodies with PBS. After washing with PBS, tissues were incubated for 30 minutes at room temperature (37°C). The chromogen used was 3,3′-diaminobenzidine (DAB) (brown) (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). Tissue sections were lightly counterstained with hematoxylin, washed, and mounted with glass coverslips, ready for evaluation using light microscopy and photomicroscopy.