Anti mmp 13

Total proteins were extracted by RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China). The protein concentration was measured by the BCA Protein Assay Kit (Thermo Fisher, Waltham, USA). Then, equal amounts of protein were loaded on 8–12% SDS-PAGE gels and transferred onto a PVDF transfer membrane (Millipore, Massachusetts, USA). After blocking with 5% skimmed milk solution, the membranes were incubated with specific primary antibodies overnight at 4°C followed by further incubation with the secondary antibody at room temperature for 2 h. Both primary and secondary antibodies were purchased from Thermo Fisher (Waltham, USA), including anti-β-actin, anti-Nrf2, anti-HO-1, anti-p-NF-κB, anti-IL-1β, anti-TNF-α, anti-Bip, anti-calpain-2, anti-active caspase-12, anti-active caspase-3, anti-Bax, anti-Bcl-2, anti-ACAN, anti-Col-II, anti-Col-I, anti-MMP-13, anti-aggrecanase 1, and secondary antibody IgG. Finally, the protein bands were detected using the ECL Plus Reagent (Thermo Fisher, Waltham, USA), and their densitometry was analyzed with the ImageJ software version 8.0(Bio-Rad, Hercules, USA). With β-actin as control, the expression of total protein was normalized.

The primary antibodies used in this study included anti-Visfatin (PA1-1045, Pierce Scientific, Hemel Hempstead, UK), α-NFκB (SC-8008, Santa Cruz Biotech, Santa Cruz, CA, USA), and anti-MMP-13 (11365013, Thermo Scientific, Hemel Hempstead, UK). The secondary antibodies used in this study were Alexa Fluor^® 555 anti-rabbit H + L (A-21428), Alexa Fluor^® 488 anti-mouse IgG1 (A-21121), and DAPI (D3571) (Life Technologies, Warrington, UK).

The total protein was extracted from 4 WT and 4 MT Chahua chickens’ ovarian tissue randomly selected using a rapid cell lysate kit (Solarbio, Beijing, China). Protein concentration was quantified using the BCA protein quantification kit (Tiangen, Beijing, China). The proteins were separated by SDS-PAGE gel electrophoresis under denaturing and non-reducing conditions and transferred to a polyvinylidene fluoride membrane (PVDF). After the addition of a 5% bovine serum albumin/TBST solution, the membrane was agitated for 1 h. Mouse monoclonal antibody anti-MMP-13 (Thermo, New York, NY, USA, 1:1000) was added, and the membrane was agitated for 30 min, followed by overnight incubation at 4 °C. The membrane was washed 3 times with TBST for 10 min each, followed by the addition of goat anti-mouse immunoglobulin G antibody (Thermo, New York, NY, USA, 1:1000) and incubation in a shaker for 1 h. The 3 TBST washes were performed again for 10 min each. The PVDF membrane was exposed to chemiluminescence to obtain the experimental results. The intensities of the blots were quantified using ImageJ software [19 (link)].