Dapi fluoromount g clear mounting agents

Formalin-fixed and paraffin-embedded tumor samples were cut into 3.5-µm sections, deparaffinized, and antigen-retrieved using sodium citrate solution (0.01 M, PH: 6.0). Then, the sections were processed for antibody staining for immunohistochemistry (IHC) or immunofluorescence (IF). For IHC, slides were incubated with primary anti-PCNA (1:250, sc-56, Santa Cruz), anti-Foxp3 (1:100, 12,653, Cell Signaling Technology) or anti-CD8 Ab (1:200, ab203035, Abcam), and then secondary antibody HPR anti-mouse or rabbit IgG (Maxim). The slides were colored with 3, 3ʹ-Diaminobenzidine (DAB, Sigma-Aldrich) and counterstained by hematoxylin. For IF, anti-CD31 (1:250, sc-376,764, Santa Cruz), anti-CD206 (1:1000, ab64693, Abcam) and anti-F4/80 (1:250, sc-377,009, Santa Cruz) were used as primary Abs while anti-mouse IgG (H + L) (1:1000, 4408, Cell Signaling Technology) and anti-rabbit IgG (H + L) (1:1000, 4413, Cell Signaling Technology) were used as secondary Abs. The sections finally were mounted using DAPI-Fluoromount-G clear mounting agents (Southern Biotech, Birmingham, UK) and images were taken using fluorescence microscopy (Nikon, Japan). Positive cells were quantified using Image J software and expressed as the mean of the percentage of positive cells ± SD with 10 randomly selected fields per sample.