Masson staining kit

The femoral head was fixed with 4% formaldehyde (Solarbio), decalcified with 10% EDTA (Solarbio), dehydrated with gradient alcohol (SINOPHARM, Beijing, China), made transparent using xylene (SINOPHARM), embedded in paraffin (SINOPHARM), and sliced with a Leica pathological slicer (RM2016, Leica, Wetzlar, Germany). The slices were dewaxed in xylene and gradient alcohol in turn. The dewaxed sections were stained according to the HE staining kit (Solarbio) and the Masson staining kit (Solarbio) and then examined under a biomicroscopy (Olympus, Tokyo, Japan).

For better evaluate the effect of IL‐6 and IL‐6R antagonist treatment on interstitial fibrosis, bladder tissue slices were stained by Masson staining kit (Solarbio) according to the manufacturer's instructions.

Samples were decalcified, dehydrated, and embedded in paraffin wax. The method of HE was according to the published study [37 (link)]. For masson staining, the procedures were conducted according to the commercially available protocol of Masson staining kit (Solarbio, China). Number of nucleus infiltrated in joint capsule was obtained in area of 25 μm*25 μm with 6 random measures to get a mean number by ImageJ 7.0 (NIH, Bethesda, MD, USA). The thickness, also obtained by 6 measures in each sample, was normalized to the value of group NS. Immunofluorescent staining in vivo and in vitro were published elsewhere [29 (link), 38 (link)]. Images were captured by microscopically (Olympus, X71).
In terms of shoulder samples used for liposome tracing, slices were blocked in 5% BSA with 0.5% Triton-X-100 (Solarbio, Beijing, China) for 60 min. Slices were then stained Dapi and observed immunofluorescently.