Detection of proliferating cell nuclear antigen (PCNA) protein expression by immunohistochemistry

Immunohistochemical staining was performed to detect the expression of PCNA in the xenograft tumors. Paraffin sections were rehydrated. The sections were subsequently treated as follows: Microwave antigen retrieval (700 W for 8 min; twice in 10 mM sodium citrate; pH 6.0) was followed by incubation with 3% hydrogen peroxide to block endogenous peroxidase and 10% goat serum (cat. no. 5560-0007; Seracare Life Sciences, Inc.) at 4˚C for 30 min to block nonspecific binding. PCNA was detected with rabbit anti-PCNA (1:100; cat. no. ab18197; Abcam) overnight at 4˚C and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:400; cat. no. ab205718; Abcam) for 1 h at room temperature. After that, 3,3-diaminobenzidine tetrahydrochloride (DAB; cat. no. 30015; Biotium, Inc.) was used for the chromomeric reaction, and hematoxylin was used to stain the nucleus for 5 min at room temperature. The staining of the tumor nuclei in each group was observed using a Leica DFC300 FX light microscope (Leica Microsystems Inc.; IL; magnification, x400). PCNA-positive cells, displaying brown-yellow granules in the nuclei, were observed in five randomly-selected high-power fields. The integrated optical density (IOD) values of the images were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.). For the negative control, the primary antibody was replaced by normal rabbit IgG.