Anti rabbit hrp dab system

Sections were undergone antigen retrieval with sodium citrate buffer and blocked by 3% goat serum (16210064, Gibco, USA) for 1 h. Then primary antibodies (Rabbit anti-CD31, 1:200, GTX130274, Gene Tex; Rabbit anti-Ki-67, 1:300, 12202 T, Cell Signaling Technology; Rabbit anti-CD163, 1:300, ab182422, Abcam) diluted in blocking buffer were used to incubate at 4 °C overnight. After washing with PBS, the Cell and Tissue Staining Kit (CTS005, Anti-Rabbit HRP-DAB System, R&D Systems) was used to detect the positive staining area. Images were taken by a microscope and analyzed by Image J software.

Paraffin sections of each group were undergone antigen retrieval with sodium citrate buffer and blocked by 3% goat serum (16210064, Gibco, USA) for 1 h. Diluted primary antibodies (anti-CD31 antibody (Abcam, ab28364); anti-Ki-67 antibody (Cell Signaling Technology, 12202T); anti-CD163 antibody (Abcam, ab182422); anti-CD86 antibody (Cell Signaling Technology, 19589S)) with blocking buffer were used to incubate at 4 °C overnight. After washing with PBS, the cell and tissue staining kit (CTS005, Anti-Rabbit HRP-DAB System, R&D Systems) was used to detect the staining. Images were taken by an inverted fluorescence microscope (Axio observer A1, Zeiss, Germany) and analyzed by ImageJ software.