Hluga180su02

The tissue chip (project no. HLugA180Su02; Shanghai Outdo Biotech Co., Ltd.) was baked in an oven at 63°C for 1 h, followed by dewaxing in an automatic dyeing machine (ST5015; Leica Microsystems). Antigens were retrieved by incubation with 0.1 M citric acid at high pressure (50 kPa) at 110°C for 5 min. The chip was blocked with 5% BSA at room temperature for 20 min and rinsed with PBS three times. The primary antibody B7H5 (cat. no. 54979T; 1:300 dilution; Cell Signaling Technology, Inc.) was added dropwise and the array was incubated at room temperature overnight at 4°C. Next, the slide was rinsed with PBS three times, the secondary antibody, anti-rabbit IgG HRP-linked antibody (cat. no. 7074S; 1:2,000 dilution; Cell Signaling Technology, Inc.), was added dropwise, and the slide was incubated at room temperature for 30 min. Finally, 3,3′-diaminobenzidine solution was added dropwise and the color development intensity was observed. Thereafter, the slide was rinsed with tap water for 5 min and Hastelloy Hematoxylin (Sigma-Aldrich; Merck KGaA) was added dropwise onto the slide, followed by incubation for 1 min. The sample was then submerged in 0.25% hydrochloric acid alcohol for no less than 2 sec, rinsed with tap water for >2 min, dried at room temperature and mounted for a light microscopic (Olympus Corporation) observation.