Horseradish peroxidase hrp labeled goat anti rabbit igg

Soybean powder (contain ∼52% protein and 1% fat), gelatin (from porcine skin, Type A, gel strength 300), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), insulin, dexamethasone, methyl isobutyl xanthine, WST-1 reagent, Safranin-O, Nile Red, and chemiluminescence HRP subtract were obtained from Sigma-Aldrich (St. Louis, MO, United States). Calcein AM, ethidium homodimer-1 (EthD-1), Hoechst 33,342, Alexa Fluor 488 Phalloidin, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and horse serum (HS) were obtained from Thermo Fisher Scientific (San Jose, CA, United States). C2C12 mouse myoblasts and 3T3-L1 mouse pre-adipocytes were purchased from Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan). Primary antibodies against myosin (MF20), myogenin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), peroxisome proliferator-activated receptor γ (PPARγ), and secondary antibodies, including goat anti-mouse IgG and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG were purchased from Thermo Fisher Scientific (San Jose, CA, United States). The fluorescent microscope and confocal microscope were obtained from Leica (Wetzlar, Germany), the fluorescence CCD imager was from GE Healthcare Life Sciences (MA, United States), and scanning electron microscope (SEM) was from Hitachi High-Tech Corporation (Tokyo, Japan).

The mouse cartilage tissues were decalcified in 10% disodium ethylene diamine tetraacetic acid (Biosharp, Hefei, China), embedded in paraffin and cut into 5-µm-thick sections. All tissues were stained with safranin O-fast green. The articular cartilage destruction was evaluated using the Osteoarthritis Research Society International (OARSI) scoring system by estimating the highest observed scores based on a previous study (Glasson et al. 2010 (link)). For immunohistochemistry (IHC), the sections embedded in paraffin were dewaxed, hydrated, and inoculated with 3% hydrogen peroxide. Thereafter, the sections were blocked with 1% bovine serum albumin (Sangon, Shanghai, China) for 15 min at room temperature and incubated with the primary antibodies against CHMP5 (1:50), collagen II (1:50) and matrix metallopeptidase 13 (MMP13; 1:50) purchased from Affinity Biosciences (Changzhou, China) at 4 °C overnight. Then, the slides were inoculated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:500; Thermo Fisher Scientific, Shanghai, China) at 37 °C for 1 h. Next, the 3, 3-diaminobenzidine tetrahydrochloride (Maixin, Fuzhou, China) was used as a chromogenic agent. The images were captured with a microscope (Olympus, Japan).