Hrp streptavidin

Hearts fixed in 4% paraformaldehyde were embedded in paraffin and then cut into 5 μm sections. Sections were incubated with an anti-HMGB1 rat polyclonal primary antibody (1:200 dilution, 60 min at room temperature; Abcam, USA) and a secondary antibody (1:100 dilution, 60 min at room temperature; Zhongshan Biotechnology, China). The sections were incubated in HRP-streptavidin (1:100; Zhongshan Biotechnology, China) for 30 min at 37°C, and the color reaction was visualized with diaminobenzidine (DAB; ZSGB-BIO, Beijing, China). For each section, five fields were randomly selected from the infarcted area of the left ventricle and photographed with a digital camera (AF6000, Leica, Germany). The mean density of HMGB1 expression in the myocardium was analyzed using Image J.

At 3 months after the BCCAO or sham surgery, the mice were anaesthetized and intracardially perfused with phosphate‐buffered saline (PBS), followed by 4% paraformaldehyde. The brains were removed, post‐fixed overnight in 4% paraformaldehyde at 4°C, and stored in 30% sucrose in 0.1 mol/L PBS (pH 7.4). Serial coronal sections (30 μm) that spanned from the anterior of the corpus callosum (bregma 0.26 mm) to the anterior of the hippocampus (bregma 0.94 mm; adjusted according to the mouse brain atlas)³¹ were cut using a cryostat. Coronal sections were treated with 3% H₂O₂ in 0.01 mol/L PBS and pre‐incubated in 5% normal donkey serum. Subsequently, the sections were incubated in a primary Ab solution overnight at 4°C. After washing, the samples were incubated in a secondary Ab solution containing donkey anti‐rabbit or donkey anti‐goat biotinylated IgG (1:200, Zhongshan Biotechnology) for 1 hour at RT. Finally, the sections were incubated in HRP‐streptavidin (1:200, Zhongshan Biotechnology) for 1 hour at RT, and the colour was developed using the conventional reaction of diaminobenzidine with H₂O₂. All images were acquired using fluorescence microscopy (Olympus IX70) or light microscopy (Olympus).

For immunohistochemistry, the paraffin-embedded sections (5 μm) were rehydrated and microwaved for antigen retrieval with EDTA and pretreated with 0.3% H₂O₂. Subsequently, the sections were blocked with goat serum and were incubated overnight at 4°C in a humidified box with specific antibodies against TLR-4 (1: 200, Abcam, USA), MyD88 (1:200, Abcam, USA), P65 subunit of NF-κB (1: 500, Abcam, USA). Then, sections were incubated in a secondary antibody (1: 100, Zhongshan Biotechnology, China) solution for 2 h at room temperature. Finally, the sections were incubated in HRP-streptavidin (1:100, Zhongshan Biotechnology, China) for 30 min at 37°C, and the color reaction was developed with diaminobenzidine (DAB). For each section, five fields were randomly chosen from the surrounding infarction areas of the left ventricle and captured with a digital camera (SZ-40, Olympus, Tokyo, Japan).