Mab 0567

For IHC, 4-µm paraffin-embedded sections were baked at 60 °C for 1 h, deparaffinized with xylene, rehydrated with a series of graded alcohols, and microwaved in EDTA antigen retrieval buffer. Then, the sections were blocked with 10% goat serum before incubation with a primary antibody at 4 °C overnight, followed by HRP-conjugated secondary antibody incubation for 30 min at room temperature. DAB was added to detect antibody binding. Once brown color appeared, the sections were immersed in distilled water to stop the reaction. The sections were counterstained with hematoxylin, dehydrated in graded alcohols and mounted. The primary antibodies were rabbit anti-human DSG2 monoclonal antibody (ab150372, Abcam, Britain) and mouse anti-human D2–40 monoclonal antibody (MAB-0567, MXB, China). The DSG2 staining results were scored based on the following criteria: (i) percentage of positive tumor cells in the tumor tissue: 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–70%) and 4 (71–100%); and (ii) staining intensity: 0 (none), 1 (weak), 2 (moderate), and 3 (strong). The staining index was calculated as the staining intensity score × the proportion of positive tumor cells (range from 0 to 12). The staining score of 6 was defined as the cutoff. Thus, patients with different positive staining levels of DSG2 expression were divided into low- and high-staining groups.