Ab243903

For immunofluorescence staining, the slides are deparaffinized and rehydrated with graded ethanol and the process of antigen retrieval and non-specific binding blocking is the same as the previous IHC.
For the multiplexed immunofluorescence of CD4, CD8, PD-1, TIM3, TIGIT, and DAPI (for nuclear staining) in the FFPE tissue, we used a validated multiple fluorescence staining kit from Servicebio (Wuhan, China), according to the manufacturer’s recommendation. The following primary antibodies were used: mouse anti-human PD-1 (Abcam, ab52587, 1:200), rabbit anti-human TIGIT (Abcam, ab243903, 1:200), rabbit anti-human TIM3 (Abcam, ab185703, 1:400), and rabbit anti-human CD8 (Abcam, ab4055, 1:200). After the slides are incubated with each primary antibody and the corresponding HRP-labeled secondary antibody, the slides need to be labeled with different fluorescent dyes. The sample scanning, spectral unmixing, and quantification of signals were conducted with the panoramic MIDI Pathology Imaging System, using the CaseViewer V.2.3 software of 3DHISTECH.

TIGIT expression in tumor tissue and adjacent normal tissue was detected by IHC. Briefly, tissue sections were deparaffinized, hydrated, and antigen retrieved with a citrate buffer (pH 6.0). After washing with PBS buffer and blocking with 10% goat serum, 70 μL anti-TIGIT primary antibody (ab243903; 1:200; Abcam) was added, and the mixture was incubated at 37°C for 1 h. Then, a secondary antibody included in the Dako K5007 kit was added. The mixture was incubated at room temperature for 30 min and rinsed with PBS buffer. Afterwards, the sections were incubated with 3.3’-diaminobenzidine (DAB), counterstained with hematoxylin, rinsed with running water and differentiated with 0.5% hydrochloric acid alcohol for 3–5 sec. Then, the sections were washed again with running water for 15 min, dehydrated, and mounted, and finally, were observed under an inverted optical microscope (Olympus, XDS-1A).

Total protein was extracted from the cell lines using RIPA lysis buffer (Beyotime, Shanghai, China). The protein content was evaluated by BCA method using a commercial kit purchased from Bioteke (Beijing, China). Protein (20 μg) was separated on 10% SDS-PAGE gels, and then was transferred onto PVDF membranes (Millipore, Bedford, USA) and blocked with 5% skimmed milk under room temperature. Subsequently, rabbit monoclonal primary antibodies against TIGIT (ab243903; 1:4000; Abcam) and against α-tubulin (66,031; 1:10,000; Proteintech, China) were added and incubated overnight at 4°C. PVDF membrane was washed four times with TBST for 5 min each time and then incubated with goat anti-mouse or anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. After washing PVDF membrane for four times, immunoreactive bands were displayed by enhanced chemiluminescence (ECL), photographed in grayscale and quantified using the Image J program (NIH, Bethesda, MD, USA). The α-tubulin was used as an internal reference to normalize bands. The ratio of IOD TIGIT/IOD α-tubulin indicated the relative expression of TIGIT protein.