PCNA Immunohistochemistry Protocol

Tumors were fixed with 10% neutral buffered formalin for 48 h, paraffin-embedded, and cut in 5-μm sections. Heat-induced antigen retrieval was performed in citrate buffer, and after blocking endogenous peroxidase, slides were incubated with primary antibody anti-PCNA antibody (PC10) (1:200 dilution, sc-56, Santa Cruz Biotechnology) as previously indicated [26 (link)]. Then, slides were incubated with a biotinylated antibody and developed with the ABC-HRP Kit (Vector Laboratories, Burlingame, CA, USA) and peroxidase substrate DAB (Sigma-Aldrich, St. Louis, MO, USA). Afterward, slices were examined with a Zeiss Axioplan microscope equipped with a Nikon DXM1200F digital camera. The PCNA cell count was quantified in four randomly selected fields from each animal and analyzed using ImageJ 1.54e software.