Ab131044

At first, the cell or tissue protein were extracted using the RIPA lysis buffer (P0013B, Beyotime). The protein concentration was determined by the BCA protein assay kit. All samples were prepared at a final concentration of 1 μg/μl in loading buffer (CW0027S, CwBio). Protein samples (20 μg) were loaded into a 10–15% SDS-polyacrylamide gel in the Bio-Rad Electrophoresis System to separate the proteins with different molecular weight. Then, the proteins in the gel were transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% bovine serum albumin (BSA) solution, the membranes were incubated with primary antibodies (Integrin-α_L, ab186873, Abcam; Integrin-β₂, ab131044, Abcam; C1q, ab71940, Abcam; Integrin-β₃, ab75872, Abcam; CD11b, ab133357, Abcam; CD45, ab10558, Abcam; cleaved caspase-3, #9661s, Cell signaling technology; CD44, ab189524, Abcam; HO-1, ab68477, Abcam; β-tubulin, CW0098, CwBio; iNOS, ab15323, Abcam; CD206, ab125028, Abcam; Arginase-1, 93668s, Cell signaling technology; Histone H3, #9715, Cell signaling technology; GM130, 610,822, BD Biosciences) at 4 °C overnight. After incubation with the corresponding secondary antibodies, the PVDF membranes were imaged using Western chemiluminescent horseradish peroxidase (HRP) substrate (Millipore) with an imaging system (Tanon 4600, Shanghai).

After following the hydration protocol described above, the tissues were subjected to heated antigen retrieval in citrate buffer (pH 6.0), blocking in BLOXALL (30 min) and 10% goat serum in PBS (20 min) obtained from Vector Labs (Burlingame, CA), and detection of rabbit anti-ITGB2 (1:100, 4°C, overnight) (Abcam, ab131044; Cambridge, MA) using ImmPRESS^TMHRP anti-rabbit IgG and ImmPACT DAB Peroxidase kits from Vector Labs. The process was repeated for the detection of mouse anti-ICAM-3 (1:1000, 4°C, overnight) (Abcam, ab10804; Cambridge, MA), employing ImmPRESS^TMHRP anti-mouse IgG (clone ab10804) and ImmPACT SG Peroxidase kits from Vector Labs. Tissues were also stained with and without primary and secondary antibodies for both antigens to serve as staining controls.