Quantification of PCNA and MVD in Tumor Tissues

Tumor tissue sections (5 μm) were prepared for immunohistochemistry following reports [38 (link)]. Mouse anti-PCNA monoclonal antibody (1:100) (Leica Biosystems, UK) or goat anti-mouse CD31 polyclonal antibody (1:100) (Santa Cruz, USA) were used as primary antibodies. PBS instead of primary antibody was used as a negative control. The intensity of PCNA immunostaining was quantified using a computerized imaging system with 5 randomly selected fields at 200 times magnification. Staining was considered negative when the percentage of cells positive for PCNA staining was less than 10%. For MVD quantitation, 5 fields containing CD31 staining were selected and the number of CD31 positive cells in a field was quantified.