Horseradish peroxidase conjugated igg secondary antibody

Western blot was performed according to the previous study (29 (link)). The proteins were extracted from the striatum and the concentrations were determined by the BCA Protein Assay kit (Thermo Scientific, United States). The protein (30 μg) was loaded onto 12% SDS-PAGE (Solarbio, Beijing, China) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, United States). The blot was incubated in blocking buffer (5% milk in Tris-buffered saline with 0.1% Tween-20) and then incubated with rabbit anti-MBP (Cat# ab40390, Abcam, United States; 1:1,000) or rabbit anti–β-actin (1:1,000, Catalog No. 4970S; Cell Signaling Tech) overnight at 4 °C. After washing with TBST (0.01% Tween 20 in TBS), the blots were incubated with horseradish peroxidase-conjugated IgG secondary antibody (Biosharp, BL003A, 1:10,000) for 1 h at room temperature and then reacted with BeyoECL Star (Beyotime, Shanghai, China). The chemiluminescence results were recorded by Amersham Imager 680 imaging system (CTL, United States) and analyzed by ImageJ (NIH, United States).

Immunohistochemistry (IHC) analysis was performed on 5‐μm paraffin‐embedded tissue sections that were rehydrated in a graded series of ethanol (99%‐70%) and finally in distilled water. For ITGB3 staining, blocking of non‐specific antibody binding was blocked with 3% bovine serum albumin (BSA) in Tris‐buffered saline supplemented with 0.05% Tween 20 (TBST). A primary antibody against rabbit ITGB3 (ab218435, 1:100; Abcam) was diluted in 1% BSA in TBST. The antigen‐antibody complex was visualized using a horseradish peroxidase‐conjugated IgG secondary antibody (1:200; Beyotime) followed by 3,3′‐diaminobenzidine (DAB). ITGB3 immunostaining was scored as follows: score 0, no expression; score 1, weak expression; score 2, moderate expression; score 3, strong expression.29

Tissues of inflamed joints were separated and homogenized on ice in cold lysis buffer (Beyotime, China) plus 1:100 volume of phenylmethyl sulfonylfluoride (PMSF). RAW 264.7 cells were treated with LPS (1 μg/ml) and Rg1 (12.5 μM, 25 μM, and 50 μM) for 24 h and then washed with cold PBS and lysed in ice-cold lysis buffer (Beyotime, China) plus 1:100 volume of PMSF. The supernatant was aliquoted and protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Proteins were segregated by electro-blotted and SDS-PAGE into a membrane of nitrocellulose. The blots were probed with antibodies against PPAR-γ, IκBα, p-IκBα, NF-κB p65, p-p65 (all antibodies were diluted by 1:1000) at 4°C overnight and later incubated with horseradish peroxidase-conjugated IgG secondary antibody (Beyotime, China; 1:5000 dilution) for 1 h. The expression of every protein was detected by the detection system of ECL (ChemiDoc^™XRS, Bio-Rad, Shanghai, China). The respective bands were quantitated and images were collected utilizing Quantity One Software (Bio-Rad). The fold increase over control was used to express the results [42 (link)].