Rabbit anti collagen 1 antibody

Cardiac tissues were fixed in 10% buffered formalin for 30 min, dehydrated overnight in 75% ethanol, and embedded in paraffin. Serial sections (4 μm) were cut for morphometric analysis of the atherosclerotic lesions. The sections were stained with H&E, Masson’s trichrome, and Periodic Acid-Schiff (PAS) for histological analysis. For immunohistochemical staining, the heart sections were deparaffinized and rehydrated. Next, the sections were blocked with 3% H₂O₂ in methanol for 15 min to inactivate the endogenous peroxidases and incubated overnight at 4 ℃ with the primary antibodies: TGF-β (rabbit anti-TGF-β antibody, 1:300; Proteintech, Wuhan, China), collagen I (rabbit anti-collagen I antibody, 1:1000; Proteintech), collagen III (rabbit anti-collagen III antibody, 1:1000; Proteintech), MMP2 (rabbit anti- MMP2 antibody, 1:200; Proteintech), MMP9 (rabbit anti- MMP9 antibody, 1:300; Proteintech), LOX-1 (rabbit anti-LOX-1 antibody, 1:300; Abcam, England, CD36 (rabbit anti-CD36 antibody, 1:500; Proteintech). The sections were then incubated for 30 min at room temperature with a goat anti-rabbit HRP secondary antibody (anti-rabbit Universal Immunohistochemical Detection Kit; Proteintech). All sections were examined with an Olympus B×40 upright light microscope (Olympus, Tokyo, Japan).

The cardiac tissues were harvested, and protein extracts prepared, according to established methods. Extracts were separated in sodium dodecyl sulphate–polyacrylamide electrophoresis gels (8–15%) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% milk and then incubated with indicated primary antibodies at 4 °C overnight. Primary antibodies against TGF-β (rabbit anti-TGF-β antibody, 1 : 1000; Proteintech, Wuhan, China), Smad3 (rabbit anti-Smad antibody, 1 : 1000; Proteintech), collagen I (rabbit anti-collagen I antibody, 1 : 1000; Proteintech), collagen III (rabbit anti-collagen III antibody, 1 : 1000; Proteintech), α-SMA (rabbit anti-α-SMA antibody, 1 : 1000; Proteintech), P53 (rabbit anti-P53 antibody, 1 : 1000; Proteintech), bcl-2(rabbit anti-bcl-2 antibody, 1 : 1000; Proteintech), anti-β-actin (1 : 1000; Cell Signaling Technology). After washing, the membranes were incubated with the appropriate secondary antibody (anti-rabbit Ig-G, 1 : 1000; Cell Signaling Technology) for 1 hour. This analysis was carried out independently three times. Protein levels were expressed as protein/β-actin ratios to minimise loading differences. The relative signal intensity was quantified using NIH ImageJ software.