Anti orm1

Mouse hearts were fixed in 4% paraformaldehyde (pH 7.4) overnight, embedded in paraffin, and continuously sectioned at a thickness of 5 μm. For dewaxing, sections were sealed with phosphate-buffered saline (PBS) containing 5% normal target serum and 1% bovine serum albumin (BSA) and then incubated overnight at 4°C under humidified conditions with anti-ORM1 (Abcam, UK), anti-Nrf2 (Abcam, UK), anti-HO-1 (Abcam, UK), anti-4-HNE (Abcam, UK), and anticleaved caspase-3 (Cell Signaling Technology, USA). A routine histological examination was performed using light microscopy. Images were examined with a Zeiss Axio Imager 2 microscope (Zeiss, Germany).

After collecting cells and heart samples from the different treatment groups and extracting proteins, the protein concentrations were determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific, USA). The samples from each group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked by incubation with 5% skimmed milk (containing 0.1% Tween-20) for 2 h at room temperature. After incubation, the membrane was further incubated with the primary antibodies anti-ORM1 (Abcam, UK), anti-Nrf2 (Abcam, UK), anti-HO-1 (Abcam, UK), anti-4-HNE (Abcam, UK), and anticleaved caspase-3 (Cell Signaling Technology, USA). Next, the membrane was washed in Tris-buffered saline Tween (TBST) and incubated with the corresponding secondary antibody (Abcam; at a dilution of 1 : 5000) for 2 h at room temperature. The Amersham Imager 680 (GE, USA) was used to detect the bands, and the signal was quantified.