Df13312

The sections were repaired with an EDTA antigen repair buffer (pH = 8, G1206, Servicebio, CHN) in a microwave, and the 3% BSA was added dropwise to block for 30 min. Then the CD45R (1 : 50, sc19597, Santa, US), INSR-β antibody (1 : 50, sc57342, Santa, US), CXCL16 (1 : 100, DF13312, Affinity, CHN), anti-LDL receptor antibody (1 : 100, ab30532, Abcam, US) and anti-TF antibody (F-8) (1 : 50, sc-373785, SCBS, US) were used for incubating with the sections overnight at 4°C, and then after washing, the tissue that was incubated with the goat antirabbit IgG H&L (ab150078, Abcam, US) that was incubated in dark for 50 min. Following PBS washing, the nuclei were counterstained with DAPI (G1012, ServiceBio, CHN). Thirdly, after being washed, the sections were quenched by the antifluorescence quenching sealing reagent (G1401, Servicebio, CHN) for 5 min and rinsed with running water for 10 min. Between every two steps, the sections were washed 3 times with PBS (pH = 7.4) on the shaking table. Finally, a NIKON eclipse upright microscope was used to observe and image the fluorescence (the nucleus is blue, and the positive expression is red or green).

The tissue was soaked in RIPA lysate and homogenized on ice; then it was centrifuged. The total protein concentration was measured by the BCA Protein Assay Kit (PC0020, Solarbio, CHN) and mixed with a loading buffer (1 : 4) in a 100°C water bath for 5 min. The SDS-PAGE and PVDF membranes were used to separate the protein by vertical electrophoresis and wet transfer. Following blocking by 5% BSA, the sample was incubated with the primary antibody for one night. Secondarily, the sample was incubated with HRP-conjugated goat IgG secondary antibodies at 20°C for 1 h. After wet transfer, the sample was cleaned 3 times/10 min with TBST between every two steps. Finally, ECL luminescent reagent was used to present the photo in the chemiluminescence imager. The antibodies for CXCL16 (1 : 2000, DF13312) and GAPDH (1 : 5000, AF7021) were purchased from Affinity Biosciences, Ltd. (Jiangsu, CHN). And NF-κB (1 : 1000, 6956T), LC3 (1 : 1000, 4599T), and P62 (1 : 1000, 5114T) antibodies were provided by Cell Signaling Technology, Inc. (MA, US). The TF antibody (1 : 100, sc-373785) was bought from Santa Cruz Biotechnology, CA (MO, US).

Immunofluorescence staining was performed on tissue and cell samples. For tissue samples, slides were deparaffinized, rehydrated, and treated with sodium citrate buffer for antigen retrieval. Slides were incubated with 3% goat serum for 30 min followed by primary antibodies: CXCL16 antibody (DF13312, Affinity Biosciences, China), VCAM1 antibody (DF6082, Affinity Biosciences, China), SELE/CD62E antibody (DF6914, Affinity Biosciences, China), IL-23A antibody (DF13760, Affinity Biosciences, China), IL-17A Polyclonal antibody (26163-1-AP, PTG, China), Purified anti-human/mouse CD3ε (362701, Biolegend, USA), CD31 antibody (ab134168, Abcam, USA), Anti-E Cadherin antibody (ab231303, Abcam, USA). The secondary antibody used were Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (A-11008, Invitrogen), Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (A-11005, Invitrogen).