Immunohistochemical Analysis of OLFM4, PCNA, Lysozyme, and BMI1

Immunohistochemistry (IHC) was performed as previously described [25 (link)]. Briefly, the 4-μm thick sections were deparaffinized in xylene, rehydrated in graded alcohol, and washed with 0.01-M PBS (pH 7.4). After the epitope retrieval with a citrate buffer (pH 6.0; Dako, Carpinteria, CA, USA) and the blocking with a blocking solution (Dako), the tissue sections were incubated with anti-OLFM4 (1:400 dilution, Cell Signalling Technology, Danvers, MA, USA), anti-PCNA (1:100 dilution, Abcam, Cambridge, UK), anti-lysozyme (1:3000 dilution, Abcam), and anti-BMI1 (1:400 dilution, Abcam) antibodies overnight at 4 °C. After washing with PBS, the sections were incubated for 30 min with horseradish peroxidase-conjugated secondary antibodies (Dako) and the antigen–antibody interaction was visualized using the chromogenic substrate 3,3′ diaminobezidine (DAB; Dako).