Alexa fluor 549 mouse anti cd3

Tissue microarrays (HRec-Ade180Sur-03, Shanghai Outdo Biotech Co.) were constructed using formalin-fixed, paraffin-embedded carcinoma tissues matched to adjacent normal tissues collected from 90 colorectal cancer cases (cohort 2). Immunofluorescence was performed following an established protocol. Briefly, tissue microarrays were dewaxed, rehydrated through graded ethanol, and incubated with 3% hydrogen peroxide for 30 mins. Antigen retrieval was performed by heating the sections to 95 °C in 0.01 M citrate buffer (pH6.0) for 15 mins. Slides were then washed in PBS for 15 mins, treated with 10% normal horse serum for 30 mins and incubated with the primary antibodies, including Alexa Fluor 549 mouse anti-CD3 (BioLegend, San Diego, USA) and DyLight 488rabbit anti-TIGIT (BioLegend, San Diego, USA). After staining, the slides were mounted with ProLong Gold Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher, USA). The images were taken using the Leica Aperio System (Leica, USA).