Hrp labeled goat anti rabbit igg

Staphylococcus aureus cultures were treated with various echinatin concentrations (0 to 32 μg/mL) and incubated with shaking until the late logarithmic growth period was reached. The supernatant was then collected and mixed with protein loading buffer for 10% SDS–PAGE. Proteins were then transferred onto PVDF membranes by a semidry transfer device. The membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature and then washed three times with PBST. The membrane was then incubated with the primary rabbit anti-Hla antibody (1:3000, Sigma–Aldrich) followed by incubation with the HRP-labeled goat anti-rabbit IgG (1:5000, Sigma–Aldrich) secondary antibody. After washing the membrane three times, the bands were observed by a chemiluminescence detection device after adding BeyoECL Star reagent (Beyotime, Beijing, China).

Myocardium tissues were fixed in 10% Neutral buffer formalin and then embedded in OCT and cut into 6-μm-thick slices. After blocking with 3% hydrogen peroxide solution for 10 min, the sections were subsequently incubated overnight at 4 °C with the primary antibody (rabbit anti-mouse SIRT7, 1:200, ab78977, Abcam). Sections were then incubated with HRP-labeled goat anti-rabbit IgG (1:1000, Sigma) at 37 °C for 15 min. After three times of washing with PBS, the sections were stained with diaminobenzidine, and observed under an invert fluorescence microscope (Olympus Ckx53).

Different concentrations of 2,3-DHKV (0–32 μg/ml) were added to the suspension of S. aureus with an initial OD₆₀₀ of 0.3 and incubated with shaking until late logarithmic growth. Subsequently, the supernatant was collected and immediately subjected to 12% SDS-PAGE, followed by the proteins was transferred to the PVDF membrane via a semi-dry transfer apparatus. The membranes were blocked for 2 h at room temperature with 5% bovine serum albumin (BSA), followed by three washes with PBST. Next, the protein membranes were immunoreacted with rabbit anti-Hla antibody (1:3000, Sigma-Aldrich) and HRP labeled goat anti-rabbit IgG (1:800, Sigma-Aldrich), respectively. After washing the membrane for 3 times, Super ECL Plus (S6009M, US EVERBRIGHT Suzhou, China) was added to visualize the bands using an enhanced chemiluminescence (ECL) detection device.