Nb100 56566

Western blot was used to determine the protein levels of TLR4, as described previously 15. Heart tissue was homogenized and isolated cardiomyocytes were lysed in RIPA buffer supplemented with protease inhibitors (Beyotime Institute of Biotechnology, Jiangsu, China), sonicated on ice and protein concentration was determined using a bicinchoninic acid kit (Beyotime Institute of Biotechnology). The lysates (20 μg of total proteins) were electrophoresed on a 10% SDS‐PAGE gel and transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively. The signal was visualized using chemiluminescence reagents, scanned with a GeneGnome Syngene Bio Imaging system and quantified by densitometry.

Heart tissue and cultured cardiomyocytes were lysed in RIPA buffer containing protease inhibitors at 4°C. Lysates were collected and determined with protein concentration using a bicinchoninic acid kit. For Western blot, 20 µg of total proteins was separated on the SDS‐PAGE gel and transferred onto PVDF membrane (Immobilon‐P Transfer Membrane, Millipore Corp). The membrane was then blocked with PBST buffer containing 5% non‐fat dried milk for 1 hour and further incubated overnight at 4°C with primary antibodies against TLR3 (ab62566, Abcam), TLR4 (NB100‐56566, NOVUS) or FOXC1 (#8758, Cell Signaling Technology). After that, the membrane was incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (sc‐2004, sc‐2005, Santa Cruz Biotechnology) for 1.5 hours at room temperature and visualized by chemiluminescence reagents. GAPDH (ab8245, Abcam) was used as a loading control.

Heart samples embedded in paraffin were sectioned transversely at a thickness of 5 μm, mounted on gelatin‐coated glass slides, dried in an oven and stored at room temperature. Before staining, slides were deparaffinized/rehydrated, antigen retrieved by microwaving and blocked with 5% bovine serum albumin. Slides were then incubated overnight with primary antibodies against TLR4 (diluted 1:100, Cat. NB100‐56566; Novus Biologicals) and CD45 (diluted 1:50, Cat. ab10558; Abcam, Shanghai, China), a pan‐leucocyte marker and visualized with fluorescence‐labelled second antibodies. Similarly, isolated cardiomyocytes from sham and CHF rats were seeded on gelatine‐coated coverslips, and stained with anti‐TLR4 antibodies. Confocal microscopy was carried out using a Leica TCS SP5 microscope.