Immunohistochemical Profiling of Premalignant Tumors

At least 15 premalignant tumors per group were used to perform immunostaining experiments as described earlier [5 (link)] in Figure 1, Figure 2, Figure 3 and Figure 4. The slides were prepared for immunostaining using deparaffinization, rehydration, and antigen retrieval. Finally, immunostainings were performed using a SuperPictureTM 3rd Gen IHC detection kit (Cat#87-9673; Invitrogen, Camarillo, CA) as per the manufacturer’s instruction. Briefly, the samples were blocked for endogenous peroxidase activity followed by incubation in protein-blocking buffer for 20 min. Tissue samples were then washed and incubated overnight at 4 °C with primary antibodies including ALDH1 (Cat#ab23375, Abcam, Cambridge, MA, dilution 1:100), BMI1 (Cat#NBP18732, Novus Biologicals, Littleton, CO, dilution 1:100), CD133 (Cat#PAB12663, Abnova, Taipei, Taiwan, dilution 1:100), DCLK1 (Cat#ab37994, Abcam, dilution 1:25), LGR5 (Cat#CF503316, Origene, Rockville, MD, dilution 1:100), MSI1 (cat#ab52865, Abcam, dilution 1:100), and β-catenin (Cat#9587S, Cell Signaling Technology, Danvers, MA, dilution 1:200). After multiple washes, samples were incubated with HRP-conjugated secondary antibody for 30 min, and signals were detected using 3,3’Diaminobenzidine (DAB) chromogen. Finally, sections were counterstained with hematoxylin, followed by permanent mounting and microscopic imaging.