Af0134

IHC staining was performed using rabbit polyclonal anti-OCN (DF12303) and anti-COL I (AF0134) antibodies (Affinity Biosciences). In brief, the femoral heads were decalcified, embedded with paraffin, and sectioned at a thickness of 5 μm in the coronal plane. The sections were immersed in antigen retrieval solution (1.8% citric acid and 8.2% sodium citrate) for 10 min and then in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. After blocking in goat serum (Solarbio) for 15 min, the sections were incubated overnight at 4°C with anti-OCN or anti-COL I antibody (1 : 50), followed by incubation with horseradish peroxide-conjugated goat anti-rabbit antibody (#31460, Thermo Fisher Scientific, 1 : 500) for 60 min at 37°C. The sections were stained with DAB (Solarbio) and counterstained with hematoxylin (Solarbio). The pictures were captured using a microscope (×100) (OLUMPUS, Japan).

Each rat myocardial sample, formalin-fixed and paraffin-embedded, was cut into 5 μm sections. Slides were stained with haematoxylin and eosin (H&E) for standard histology. The distribution of collagen was carried out by Masson's trichrome staining and picrosirius red staining. The quantitative analysis of the cardiomyocyte size and collagen volume was measured with the Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA). Frozen sections were stained with Oil Red O to detect liquid content. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was utilized to assess cell death (Servicebio, Wuhan, China). After antigen retrieval in a citrate buffer (0.01 M, pH 6.0) and endogenous peroxidase blocking, slides were incubated with primary antibodies against collagen I (Affinity, AF0134, 1 : 100), collagen III (Affinity, AF0136, 1 : 100), NLRP3 (Abcam, ab210491, 1 : 200), ASC (Affinity, DF6304, 1 : 100), GSDMD (Affinity, AF4012, 1 : 100), IL-1β (Abcam, ab9722, 1 : 200), IL-18 (ProteinTech, 10663-1-AP, 1 : 300), and NEK7 (Affinity, DF4467, 1 : 100) at 4°C overnight. Thereafter, the tissues were incubated with HRP-conjugated secondary antibodies at 37°C for 30 minutes and observed with microscope (ZEISS, Axio Vert.A1, Jena, Germany).

Proteins were extracted by mixing RIPA lysate (R0010, Solarbio) and PMSF (P0100, Solarbio). The concentrations of proteins were assayed by the BCA kit (PC0020, Solarbio). According to different molecular weights, 5% concentrated gel and 8% separation gel concentrations were used in the SDS-PAGE. After transferred to a PVDF membrane, the proteins were blocked by prepared 5% (M/V) BSA (Biosharp, BS043, China) in TBST buffer for 1 h and incubated overnight at 4 °C with the following primary antibodies: α-SMA (1: 500), COL-I (1: 500, AF0134, Affinity), TGF-β1 (1: 1000, AF1027, Affinity), proliferating cell nuclear antigen (PCNA) (1: 500, A12427, Abclonal, China), p-MEK1/2 (1: 500, AP0209, Abclonal), MEK1/2 (1: 500, A4868, Abclonal), p-ERK1/2 (1: 500, AF1015, Affinity), ERK1/2 (1: 500, AF6240, Affinity) and GAPDH (1: 10000, 60004-1-Ig, Proteintech, China). Next, the membrane was incubated with goat anti-mouse IgG (1: 3000, SE131, Solarbio) or goat anti-rabbit IgG (1: 3000, SE134, Solarbio) secondary antibody for 40 min at 37 °C. At last, the specific protein bands were visualized with Western ECL Substrate (D1010, Solarbio).