Ab238151

Whole hindlimbs were resected from each animal and placed in 10% neutral buffered formalin for fixation. Hindlimbs were subsequently rinsed in saline and decalcified with Cal-EX™ solution (Catalog # CS510-1D, ThermoFisher) for 14 days. Decalcified samples were embedded in paraffin blocks for fixation and sectioning. Coronal sections were cut to a thickness of five microns and stained with hematoxylin and eosin (H&E) for histologic analysis. To evaluate synovitis, H&E sections were used to evaluate vascularity (0–2), Detritus (0–2) and Fibrosis (0–3) as previously described [15 (link)]. In addition to H&E, sections were stained for presence of osteoclasts (OC) at Day 14 and Day 56 post-injury using the OC surface marker DC-STAMP. Briefly, decalcified and rehydrated samples were blocked with 5% normal goat serum (NGS) for 1 h then incubated with DC-STAMP (Catalog # ab238151, Abcam) diluted 1:50 with NGS. Following incubation, slides were incubated in goat-anti-rabbit-HRP secondary (Catalog # ab214880, Abcam) and then developed with DAB substrate (Catalog # SK-4105, Vector labs).

The proteins were extracted using RIPA Lysis Solution (P0013 C, Beyotime, Shanghai, China) and protein concentrations were measured using a bicinchoninic acid protein assay kit (KeyGen Biotech Co., Ltd, Nanjing, China). The proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Roche, Switzerland). The membranes were incubated with the following primary antibodies: IFIT1 (ab236256, Abcam, USA), ATP6V0D2 (H00245972-M01A, Abnova, Shanghai, China), DC-STAMP (ab238151, Abcam), ATP6i (H00000525-M02, Abnova), CTSK (H00001513-M01, Abnova), TRAP (ab2721, Abcam), p-JAK1 (ab138005, Abcam), JAK1 (ab133666, Abcam), p-STAT3 (ab267373, Abcam), STAT3 (ab68153, Abcam), c-Fos (ab222699, Abcam), MMP9 (ab76003, Abcam), NFATc1 (GTX09510, GeneTex, USA), and β-actin (C1313, Applygen, Beijing, China). After a 12 h incubation, the membranes were incubated with the secondary antibody, and the intensity of protein expression was detected by ChemiScope 3300 Mini (CLINX, Shanghai, China) using enhanced chemiluminescence (Beyotime, Beijing, China).