Safranine o

After euthanasia at 4 and 8 weeks, the knee joins were fixed with 4% PFA and decalcified in ethylene diamine tetra acetic acid (EDTA, pH = 7.2, Macklin, China) by Ultrasonic Decalcifying Unit (USE 33, Germany) for 1 month. The joint samples were embedded and sectioned in paraffin (5-μm thickness) for further staining. The joint sections were respectively stained with H&E (Solarbio, China) and Safranine O-fast green (Safranine O, Solarbio, China) for histological analysis, and OA progression was evaluated according to Mankin scoring as previously described by 2 blinded observers [41 (link)]. Specifically, for immunohistochemical staining, the sections were initially incubated with rabbit polyclonal anti-IL6 or anti-MMP13 (Boster, China) antibodies overnight at 4 °C followed by binding with biotinylated secondary antibodies (ZS-Bio, China). The slides were photographed by optical microscopy (OLYMPUS BX53F, Japan).

Rats were anesthetized with 10% chloral hydrate (intraperitoneal injection), and tissues were fixed by transcardial perfusion of phosphate-buffered saline (PBS) followed by 10% formalin. Tissue was removed and fixed overnight in 10% formalin followed by a 24-h immersion in 70% alcohol. Tissue was then embedded in paraffin and sectioned (4 μm thickness). Tissue sections were deparaffinized and stained with hematoxylin and eosin (Baso, Zhuhai, China), EVG (Baso, Zhuhai, China), trichrome masson (Baso, Zhuhai, China), Alizarin Red S (Solarbio,Beijing,G3280), Safranine O (Solarbio,Beijing,G1371), or Sirius Rosa BB (Solarbio,Beijing,S8060) according to the manufacturer’s recommendations. Neointimal and adventitial thickness were measured as we previously described³⁸ . The patency of the patch angioplasty and tube graft was confirmed by observation of the histology sections.