Rabbit anti per1

Dewaxing and hydration were performed as we described previously. We used 3% hydrogen peroxide to block endogenous peroxidase activity for 15 min. And the sections were antigen-retrieved in citrate solution by microwave. Following by serum blocking for 1 h and primary antibody including rabbit anti-PER1 (Affinity, USA, 1:100), rabbit anti-MMP13 (Proteintech, China, 1:300) and rabbit anti-NF-κB p65 (Affinity, USA, 1:100) at 4 °C were incubated overnight individually. Secondary antibody was incubated for 1 h after PBS washing. Finally, we used DAB method to visualize the immunoreactive cells and hematoxylin to stain the nucleus. We used ImageJ 1.52i (National Institutes of Health, Germany) to measure the average optical density in anterior, middle and posterior fields of each section.

The collected protein samples were quantified using BCA method (Yeasen, China) and separated by 10% SDS-PAGE. Then, the protein was transferred onto the PVDF membrane (Millipore, Germany). After blocking with 5% non-fat milk at room temperature for 1 h, the membrane was incubated with primary antibodies including rabbit anti-PER1 (Affinity, USA, 1:800), rabbit anti-MMP13 (Proteintech, China, 1:1000), rabbit anti-NF-kB p65 (Affinity, USA, 1:500), rabbit anti-Phospho-NF-kB p65 (Ser536) (Affinity, USA, 1:500) and rabbit anti-GAPDH (Affinity, USA, 1:2000) at 4 °C for 16 h. Following the washing with 0.1% TBST, the secondary antibody (Beyotime, China) was incubated with the membrane. After washing with 0.1% TBST, a chemiluminescence ECL system (Millipore, Germany) was used to visualize the immunoreactive proteins.

The collected protein samples were quantified using BCA method (Yeasen, China) and separated by 10% SDS-PAGE. Then, the protein was transferred onto the PVDF membrane (Millipore, Germany). After blocking with 5% non-fat milk at room temperature for 1 h, the membrane was incubated with primary antibodies including rabbit anti-PER1 (Affinity, USA, 1:800), rabbit anti-MMP13 (Proteintech, China, 1:1000), rabbit anti-NF-kB p65 (Affinity, USA, 1:500), rabbit anti-Phospho-NF-kB p65 (Ser536) (Affinity, USA, 1:500) and rabbit anti-GAPDH (Affinity, USA, 1:2000) at 4 °C for 16 h. Following the washing with 0.1% TBST, the secondary antibody (Beyotime, China) was incubated with the membrane. After washing with 0.1% TBST, a chemiluminescence ECL system (Millipore, Germany) was used to visualize the immunoreactive proteins.