We used tissue specimens (5 μm thickness) from three patients with colonic adenocarcinoma at stage III (BioChain Institute, Inc. (Newark, CA)). Tumor tissue related data are listed in Supplemental Table S1, Supporting Information. The FFPE slides were processed as described previously [24 (link),25 (link)]. Ultimately, the slides were blocked with 2% BSA for 1 h at room temperature and incubated with a mouse anti-human ALDH1A1 monoclonal antibody (1:75 dilution, BD Transduction Laboratories, NJ) overnight at 4°C and washed with PBS three times (10 min each). Then a goat anti-mouse HRP-conjugated secondary antibody (1:200, Vector laboratories, Burlingame, CA) was used to incubate the slides for an hour. The slides were washed again with PBS three times (10 min each), and the immunoreactions (brown) were detected using the DAB Substrate kit (Vector laboratories, Burlingame, CA). Hematoxylin counterstaining was performed for nucleus visualization. The slides were then dehydrated in 70%, 95%, and 100% ethanol for 30 s each, rinsed in xylene for a few seconds, airdried in a fume hood, and then placed in a desiccator to dry completely.