Ab190377

The BV2 cells were washed once with ice-cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) for 20 min. Fixed BV2 cells were blocked and permeabilized with 1% goat serum and 0.1% Triton X-100 in PBS, followed by incubation with primary antibodies for anti-light chain 3 (LC3; 1:200; PM036, MBL, Woburn, MA, USA) and anti-HMGB1 1:200; ab190377, Abcam, Cambridge, UK) at 4 °C overnight. For the double immunostaining, FITC ant-rabbit IgG (Merck Millipore, Burlington, MA, USA) or rhodamine anti-mouse IgG (Merck Millipore) was used as the secondary antibody and incubated for 1 h at room temperature. The cells were counterstained with DAPI (Merck Millipore) to visualize the nuclei and then observed under a fluorescence microscope (Axio Observer; Zeiss, Oberkochen, Germany).

The slices were subjected to immunohistochemical staining. After the cells were fixed with 4% paraformaldehyde for 30 min, they were permeabilized with 0.3% Triton X-100 for 30 min. Then, the slices were blocked with 5% bovine serum albumin (BSA) at 37 °C for 90 min. The slices were incubated with primary antibodies against Caspase 1 (1:300, 22915-1-AP, Proteintech, USA), NLRP3 (1:300, 19771-1-AP, Proteintech, USA), HMGB1 (1:200, ab190377, Abcam, UK) and ionized calcium-binding adaptor molecule-1 (IBA-1) (1:50, 10904-1-AP, Proteintech, USA) at 4 °C overnight. The slices were incubated with secondary antibodies at 37 °C for 60 min. The slices were then preserved in glycerin buffer and observed under fluorescence microscopy.

The retina was harvested and homogenized in lysis buffer containing protease and phosphatase inhibitor mini tablets (Thermo Fisher Scientific, Waltham, MA). Nuclear expression of HMGB1 was detected in the nuclear fractions extracted by the NE‐PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher, No. 78833). The protein concentration was determined with bicinchoninic acid protein assay. Equal amounts of protein were loaded, and western blotting was performed as previously described.²⁹ The band intensities were measured using the Image J software. Primary antibodies included ZO‐1 antibody (Invitrogen, 339 100), vascular endothelial‐cadherin antibody (Cell Signaling Technology, #2158), claudin‐5 antibody (Santa Cruz, sc‐374 221), (platelet derived growth factor receptor beta) PDGFR‐β antibody (Santa Cruz, sc‐374 573), HMGB1 antibody (Abcam), RAGE antibody (Abcam, ab30381), nuclear factor kappa B (NF‐κB) antibody (Abcam, ab32536), TLR4 antibody (Abcam, ab190377), tumor necrosis factor alpha (TNF‐α) antibody (Abcam, ab183218), interleukin (IL)‐1β antibody (Abcam, ab234437), and intercellular adhesion molecule 1 (ICAM‐1) antibody (Abcam, ab222736). β‐actin antibody (Abcam, ab6276) was used as the loading control. The assays were independently repeated three times for statistical analysis.