Anti heme oxygenase 1 ho 1 antibody

The fixed samples were subsequently decalcified for 4 weeks in EDTA–glycerol solution, embedded in paraffin, and sectioned in the coronal plane at a thickness of 4 μm. After deparaffinization, hematoxylin–eosin (HE) staining and immunohistochemical staining were performed. Immunohistochemical staining samples were incubated with anti-alkaline phosphatase (ALP) antibody (Abcam, MA, USA; 1 : 2000), anti-osteocalcin (OCN) antibody (Abcam; 1 : 200), anti-osteoprotegerin (OPG) antibody (Abcam; 1 : 200), anti-RANKL antibody (Abcam; 1 : 200), anti-heme oxygenase-1 (HO-1) antibody (Abcam; 1 : 1000), anti-glutathione peroxidase (GPX) antibody (Abcam; 1 : 50), anti-superoxide dismutase 1 (SOD1) antibody (Abcam; 1 : 500), and anti-monocyte chemotactic protein-1 (MCP-1) antibody (Abcam; 1 : 500). Then, the procedures were performed following standard protocols. Quantitative analysis was performed using the ImageJ software (Rawak Software Inc., Germany).

WOC was prepared by us according to designed prescription and the China patent (No:201410564056.x). Pure walnut oil was purchased from Valder Fields CO., Ltd (Yuxi, China), and Vitamin C (VC) from Solarbio Science & Technology CO., Ltd (Beijing, China). D-gal was purchased from Sigma-Aldrich (St Louis, MO, USA). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from TCI (Shanghai) Development Co., Ltd (Shanghai, China). Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and malondialdehyde (MDA) kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). BCA protein assay kit and SDS-PAGE kits were purchased from Multi Sciences Biotech CO., Ltd (Hangzhou, China). Anti-Heme Oxygenase-1 (HO-1) antibody and anti-Klotho antibody were purchased from Abcam (Shanghai, China). Anti-iNOS antibody and anti-β-actin antibody were purchased from cell signaling technology (Danvers, USA).

Lipopolysaccharide (LPS, Escherichia coli O127:B18), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), sodium nitroprusside (SNP), Trolox, nitro blue tetrazolium (NBT), thiobarbituric acid (TBA), Dichlorodihydrofluorescein diacetate (DCFH-DA), and phenazine methosulfate (PMS) were obtained from Sigma (St. Louis, MO, USA). Anti-catalase (CAT), iNOS, COX-2, and peroxidase-conjugated secondary anti-rabbit antibodies were provided from Cell Signaling Technology (Beverly, MA, USA). Anti-IL-1β, glutathione peroxidase (GPx), superoxide dismutase-2 (SOD-2), and peroxidase-conjugated secondary anti-mouse antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-heme oxygenase-1 (HO-1) antibody was acquired from Abcam (Cambridge, UK). All other reagents were of the highest grade commercially available.

The tissues and cells were washed twice using ice-cold PBS and lysed in lysis buffer. Lysates were separated by sodium dodecyl sulfate-polyacrylamide (10%−12%) gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were washed with Tris-buffered saline/Tween-20 (TBST) and blocked in 5% skim milk powder dissolved in TBST for three hours, followed by the incubation with the respective primary antibody at dilutions according to the supplier's instructions.
The membranes were examined using an anti-NF-κB p65 antibody (1:1,000, Cell Signaling Technology), anti-heme oxygenase-1 (HO-1) antibody (1:1,000, Abcam), anti-Nrf2 antibody (1:1,000, Cell Signaling Technology), anti-Bax antibody (1:500, Santa Cruz Biotechnology), anti-cleaved caspase-3 antibody (1:500, Santa Cruz Biotechnology), anti-IL-6 antibody (1:500, Santa Cruz Biotechnology), anti-TNFα antibody (1:500, Santa Cruz Biotechnology), anti-nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) antibody (1:500, Santa Cruz Biotechnology), or anti-NQO1 antibody (1:500, Abcam). Following conjugation to horseradish peroxidase, the corresponding immunoglobulin G secondary antibody (1:1,000, Beyotime Biotechnology) was used to detect the primary antibodies. Enhanced chemiluminescence (Pierce, MA, USA) was used to visualize the bands.