Immunohistochemical Staining of PCNA and p-STAT3

Immunohistochemical staining for PCNA (sc-15402) and p-STAT3^Ser727 (sc-135649; both from Santa Cruz Biotechnology), α-amylase (cat. no. 3796) and p-STAT3^Tyr705 (cat. no. 9145; both from Cell Signaling Technology) was performed as previously described (20 (link),21 (link)). Briefly, paraffin-embedded sections (4 µm thick) were deparaffinized, rehydrated and microwave-heated for 10 min in 0.01 mol/l citrate buffer (pH 6.0) for antigen retrieval, and 3% H₂O₂ was applied to block endogenous peroxidase activity. After 15 min of incubation with blocking serum, the primary antibody or control IgG (dilution 1:50) was applied and incubated overnight at 4°C. Slides were washed three times with PBS for 5 min each time. The biotinylated secondary antibody and the streptavidin-biotin complex (Invitrogen, Carlsbad, CA, USA) were applied, each for 30 min at room temperature with an interval washing. After rinsing with PBS, the slides were immersed for 5 min in the coloring substrate 3,3′-diaminobenzidine (DAB; Sigma-Aldrich), then rinsed with distilled water, counterstained with hematoxylin, dehydrated and coverslipped.