Bs 2211r

A total cell protein extraction kit (Milipore, Billerica, MA, USA) was used to extract total protein. An equivalent amount of protein from each sample was electrophoresed by 12% SDS-PAGE and transferred to nitrocellulose membrane. After being blocked, membranes were incubated with anti-TLR4 (1:1000; PA5-23124, Invitrogen), anti- MHC-I (1:1000; ab134189 and ab22367, Abcam), anti-MHC-II (1:1000; ab157210 and ab23990, Abcam), anti-CD80 (1:1000; PA5-19211, Invitrogen and bs-2211r, Bioss, Woburn, MA, USA), anti-CD86 (1:1000; bs-1035r, Bioss), anti-TNF-α (1:1000; bs-2081R, Bioss), anti-IL-6 (1:2000; ab9324, Abcam), anti-IL-10 (1:1000; bs-0698r, Bioss), anti-CXCL10 (1:1000; PAA371Ra01; Cloud-Clone Corp., Houston, TX, USA), anti-TGF-β1 (1:1000; c0340, Assay biotechnology, Sunnyvale, CA, USA) and TGF-β2 (1:1000; 5343r-100, BioVision, Milpitas, CA, USA) overnight at 4 °C. Membranes were then washed three times with PBS/0.1%Tween-20 (5 min each), and incubated with a corresponding secondary antibody (1:5000) for 2 h at room temperature. Bands were detected using a chemiluminescence ECL kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and were quantified using the Sigma-Gel software (Jandel Scientific Software, Sari Kafael, CA, USA).

The L4–L5 spinal cord tissues of three rats in each group were selected and fixed in 10% neutral formalin, dehydrated in ethanol, embedded in wax, and then sectioned at a thickness of 4 µm. Spinal cord sections were dewaxed and incubated overnight at 4 °C with CD80 antibody (1:100, bs-2211R, Bioss, Beijing, China) and Iba-1 antibody (1:100, ab283319, Abcam, Cambridge, UK); CD206 antibody (1:100, DF4149, Affinity, Suzhou, China) and Iba-1 antibody (1:100, ab283319, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C; C3 antibody (1:100, DF13224, Affinity, Suzhou, China) and GFAP antibody (1:100, ab279290, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C; S100A10 antibody (1:100, bs-8503, Bioss, Beijing, China) and GFAP antibody (1:100, ab279290, Abcam, Cambridge, UK) were mixed and incubated overnight at 4 °C. Goat anti-rabbit IgG H&L (Alexa Flour 488,1:200, ab150077, Abcam, Cambridge, UK) and goat anti-mouse IgG H&L (Alexa Flour 647,1:200, ab150115, Abcam, Cambridge, UK) were used as secondary antibodies. Tissue sections were immunofluorescently stained and then observed under a laser confocal microscope (SP8, Leica, Wetzlar, Germany) and photographed. Fluorescence quantitative analysis was performed by Image J software.

Antibodies for IHC analysis included CRT (ab92516, Abcam), HSP90 (ab13495, Abcam), HMGB1 (ab79823, Abcam), PD-L1 (17952-1-AP, Proteintech), PD1 (18106-1-AP, Proteintech), CD11b (20991-1-AP, Proteintech), CD206(ab64693, Abcam), CD80 (BS-2211R,BIOSS), CD86 (ab213044, Abcam), MHCII (sc-59318, Santa Cruz), KI67 (ab16667, Abcam), PCNA (ab92552, Abcam), BAX (50599-2-lg, Proteintech), CASPASE3 (19677-1-AP,Proteintech), RAGE (bs-0177R) GBP5 (132201-AP, Proteintech), NF-κB (10745–1-AP, Proteintech), Phospho- NF-κB (bs-0982R, Bioss). Paraffin sections (5 μm) were dewaxed and rehydrated, antigen repaired with sodium citrate for 20 min, then incubated in 3% hydrogen peroxide for 10 min at room temperature. The paraffin sections were then blocked with 5% BSA for 30 min, stained with antibodies overnight at 4 °C, washed with PBS, and stained with secondary antibody (PV-9000, ZSGB-BIO) for 1 h at 37 °C. DAB (ZLI-9018, ZSGB-BIO) was applied for coloration for 5 min at room temperature. Hematoxylin was used to stain the nucleus.