IHC staining was performed by using the EnVision® Labeled Peroxidase System (DAKO, Carpentaria, CA, USA). All samples were fixed in 10% buffered formalin and were embedded in paraffin. Sections with 4 μm thickness were prepared, deparaffinized in xylene, rehydrated in graded alcohol, and washed with distilled water. Antigen retrieval was performed by using DAKO cytomation target retrieval solution with pH = 9, for 20 min. Internal peroxidase activity was inhibited by 3% H2O2. Tissue sections were then incubated for 30 min with the anti-VEGF antibody (mouse anti-human; DAKO Corporation – Denmark) at a 1:25 dilution. Normal samples were stained with the same amount of antibody used for staining tumor tissues. Omission of the primary antibody was employed as negative control, while pyogenic granuloma was used as positive control. Brown cytoplasmic staining for VEGF was considered as positive.
The stained slides were initially scanned at low magnification. For the slides showing heterogeneous staining, the regions with higher staining were studied. Five fields were randomly chosen, 500 cells were counted, and the percentage of staining was calculated. The extent of staining was classified as: 0 if 0–10% of tumor cells were stained, 1 if 11–25% of tumor cells were stained, 2 if 26–50% were stained, and 3 if more than 50% were stained.
The stained slides were initially scanned at low magnification. For the slides showing heterogeneous staining, the regions with higher staining were studied. Five fields were randomly chosen, 500 cells were counted, and the percentage of staining was calculated. The extent of staining was classified as: 0 if 0–10% of tumor cells were stained, 1 if 11–25% of tumor cells were stained, 2 if 26–50% were stained, and 3 if more than 50% were stained.