Rabbit anti il 6

In the present study, the following primary and secondary antibodies were used: rabbit anti-pJAK1 (Cell Signaling, Beverly, MA), rabbit anti-pSTAT3 (Cell Signaling), rabbit anti-IL-6 (Cell Signaling). For immunohistochemistry, endogenous peroxidases were quenched in 0.3% H₂O₂ in water for 10 min and then blocked with 10% normal goat serum in PBST. For detection of intracellular antigens, tissues were incubated with 100% methanol -20°C and permeabilized in blocking solution with 0.3% Triton X-100. The primary antibody in blocking buffer was applied to tissues for 60 min at room temperature or overnight at 4°C. Sections were washed in PBS and then incubated with goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA) or anti-rat biotinylated IgG (BD Biosciences, San Jose, CA). Staining was detected using Vectastain ABC kit (Vector Labs, Burlingame, CA) and sections were counterstained with hematoxylin. The area of positive area and integral optical density was calculated on the Image-pro Plus 6.0.

Protein expression was evaluated by capillary western immunoassay (JESS) using cell lysates. Total protein was extracted using Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease inhibitor cocktail. The immunoassay was performed on the JESS system (004–650, Protein Simple, a Bio-Techne Brand, San Jose, CA, USA) according to the manufacturer’s instructions using a 12–230 kDa separation module (SM-W004, Protein Simple, a Bio-Techne Brand) and an anti-rabbit detection module (DM-001, Protein Simple, a Bio-Techne Brand). The plate of the separation module was loaded and placed in the device. The reactions were run in the capillary system, the proteins were identified by specific antibodies, and their chemiluminescence reactions were measured and the digital blot images were captured. Specific antibodies used for the capillary western immunoassay were rabbit anti-MMP1 (1:50, catalog no: 54376, lot1; Cell Signaling Technology), rabbit anti-MMP3 (1:100, catalog no: 14351, lot1; Cell Signaling Technology), rabbit anti-IL-6 (1:50, catalog no: 12153, lot3; Cell Signaling Technology), and rabbit anti-β-actin (1:100, catalog no: 4970, lot15; Cell Signaling Technology).

The tissues were homogenized in a RIPA (P0013C) lysis buffer containing 1 mM PMSF (ST507). A BCA kit (P0012) was used to measure the concentration of the total protein. In this study, 20–30 ug of proteins from each sample was loaded onto a sodium dodecyl sulfate (SDS) gel for electrophoresis and then transferred to polyvinylidene difluoride (PVDF, 03010040001, Sigma, St. Louis, MI, USA) membranes. Subsequently, the blots were incubated with the primary antibodies overnight at 4 °C. On the second day, the membrane was washed and then reacted with the secondary antibody for 1 h at room temperature. Finally, the blots were visualized with an enhanced luminol-based chemiluminescent kit (34075, Thermo Fisher, Waltham, MA, USA) and scanned by an electronic camera system. The primary antibodies rabbit anti-β-actin (1:1000, 4970S), rabbit anti-IL-6 (1:1000, 12912T), rabbit anti-IL-1β (1:1000, 12703S), and rabbit anti-pP65 (1:1000, 3033S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary rabbit antibody P65 (1:1000, Ab16502) was purchased from Abcam (Cambridge, UK). The secondary antibodies goat anti-rabbit IgG (H+L) HRP (1:5000, A0218) and goat anti-mouse IgG (H+L) HRP (1:5000, A0216), RIPA, PMSF, and the BCA kit were purchased from Beyotime (Shanghai, China).