Goat anti mouse cd31 polyclonal antibody

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.

Paraffin embedded tissues were sliced in 4 μm and sections were deparaffinized in xylene followed by grated ethanol and rehydrated in PBS (pH 7.5). Then, they were microwaved for 15 min for antigen retrieval and blocked with normal goat serum for 20 min at 37 °C. The sections were incubated overnight at 4 °C with goat anti-mouse CD31 polyclonal antibody (1:50, Santa Cruz, Santa Cruz, USA), followed by washing and then incubated with rabbit anti-goat second antibody (1:50, Maixin Biotech, Fuzhou, China) at 37 °C for 30 min. The sections were stained with 3,3′-diaminobenzidine (DAB, Zhongshan Biotech Co., Ltd, Beijing, China) for 5 min and re-stained with hematoxylin for 2 min. The primary antibody was replaced by PBS in the negative controls. The stained sections were examined by microscope, and three independent fields (250 ×) were randomly selected to calculate the number of positive vessels.