Safranin o fast green

Cartilage specimens were fixed in 4% paraformaldehyde for paraffin embedding and sectioned at 5 μm. The sections were dehydrated and stained with Safranin-O/Fast green (Solarbio, Beijing, China), according to the manufacturer’s instructions. The Osteoarthritis Research Society International (OARSI) score was based on safranin O/fast green staining of each specimen, as previous described [22 (link)].

Tissue samples were fixed with 4% paraformaldehyde, paraffin‐embedded and sectioned (5 µm). After deparaffinization, sections were rehydrated and subjected to hematoxylin and eosin (HE) (Solarbio) or safranin O/fast green (Solarbio). The severity was independently scored by two researchers who did not know the group condition of the mice according to the Osteoarthritis Research Society International (OARSI) histopathology scoring system.¹⁶ Immunohistochemical staining was conducted on deparaffinized and hydrated knee joint tissue sections. After antigen retrieval, the sections were blocked, stained with anti‐MMP13 antibody (1:100) and secondary antibody. Finally, sections were stained with DAB hematoxylin.

The specimens were routinely fixed, decalcified, paraffin embedded and sectioned, and then routinely dewaxed to water. Then, the slices were stained with freshly prepared Weigert dye for 3–5 min and washed with water. The specimens were soaked in solid green staining solution for 5 min and quickly washed with weak acid solution for 10–15 s to remove the residual solid green. The specimens were added into Safranin O-fast green (Solarbio, Beijing, China) and soaked for 5 min. Dehydrated with 95% ethanol and absoluted ethanol respectively. Xylene was transparent and sealed with optical resin. Finally, the specimens were observed under microscope.

Mouse intervertebral disc tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. Tissue sections were then stained with Alcian blue (BHBT, Shanghai, China) and Safranin O fast green (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). In H&E staining, tissue sections were dewaxed with xylene I for 10 min and xylene II for 5 min, followed by rehydration with anhydrous alcohol for 1 min, 95% alcohol for 1 min, and 85% alcohol for 1 min. After that, tissue sections were stained with hematoxylin (Beyotime Biotechnology Co. Ltd., Shanghai, China) for 5 min and then with eosin (Beyotime Biotechnology Co. Ltd., Shanghai, China) for 2 min. Tissues were then dehydrated with 85% alcohol dehydration for 20 s, 95% alcohol for 1 min, absolute alcohol I for 2 min, anhydrous alcohol II for 2 min, xylene I for 2 min, and xylene II for 2 min, and sealed with neutral or Canadian balsam resin. Finally, the sections were observed under a light microscope (DMI3000, Leica, Wetzlar, Germany).

The complete knees were fixed with 4% PFA (paraformaldehyde) for 48 h, then the joints were decalcified for 2 months in a 10% EDTA (Ethylene Diamine Tetraacetie Acid, #1430, Biofroxx, Guangzhou, China) solution and paraffin-embedded. After staining the coronal sections (3 μm) with HE (hematoxylin and eosin, #C0105S, Beyotime, Shanghai, China) and SO (safranin O/Fast Green, #G1371, Solarbio, Beijing, China), the slides were examined by independent double-blinded investigators to determine the synovitis and Osteoarthritis Research Society International (OARSI) scores. To determine M1 macrophage polarization, the synovium was stained with immunofluorescence.

Clinical synovium specimens and decalcified mouse knees were fixed in 4% paraformaldehyde, followed by dehydration before embedding in paraffin to make 5‐µm sections. Sections were deparaffinized with xylene, dehydrated with gradient ethanol and then stained with H.E. (hematoxylin and eosin, G1120, Solarbio), T.B. (Toluidine bule, JL‐R4922, Jonln), or S.O. (Safranin O‐Fast Green, G1371, Solarbio). The OARSI cartilage scoring system and synovitis scores were used as previously described.^[17 (link),
⁴⁶ (link)
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