Immunohistochemical Analysis of Tumor Samples

IHC staining was performed on formalin-fixed, paraffin-embedded tumor samples and the method was as described in the previous literature^{19 (link)}. Briefly, paraffin sections were taken and dewaxed to water. Antigen repair solutions were dripped on the sections and washed with PBS 3 times. The first antibodies were added to the sections and washed with PBS (PH7.4) 3 times at 4 °C overnight. Secondary antibodies were added and rinsed with PBS 3 times again. Immunostaining was performed with DAB. The sections were counterstained with hematoxylin. The antibodies Ki67 (1:200, Abcam), CD31 (1:400, DAKO, USA), α-SMA (1:500, Themo Fisher), PD-L1 (1:600, Abcam), CD8 (1:2000, NOVUS, USA), Ly6G (1:800, Servicebio), MMP-2 (matrix metalloproteinase-2, 1:1500, Servicebio) and MMP-9 (1:800, Servicebio) were used. Visualize staining of tissue under a microscope, acquisitive and analysis image (Nikon DS-U3, Japan). The results of IHC staining were analyzed by Image J software 1.8.0 (Media Cybernetics, Rockville, MD, USA). The Ki67 and CD8 antibody staining results were evaluated by the percentage of positive cells and the positive cells density respectively, and the percentage of positive staining areas evaluated the α-SMA, CD31, PD-L1, Ly6G, MMP-2 and MMP-9 antibody staining results. Five random visual fields were counted for each sample and the average was determined.