Hematoxylin eosin

Mouse organ tissues were fixed with 4% PFA (Servicebio, Wuhan, China) and dehydrated using a graded ethanol series, followed by clearing with xylene (Shanghai, China). The pretreated tissues were embedded in paraffin (Sigma-Aldrich, USA), and once the paraffin blocks solidified, they were trimmed and sectioned into 5 μm thick slices. The paraffin sections were then deparaffinized using xylene and a graded ethanol series, stained with hematoxylin–eosin (Servicebio, Wuhan, China), and finally mounted with neutral resin (Servicebio, Wuhan, China). The morphology of the tissues was observed under a microscope, and images were captured using a slide scanner.

Fisetin powder (Cat No. HY-N0182R) purchased from MCE (Monmouth Junction, NJ, USA). LPS (Cat No. SMB00704) and Dimethyl Sulfoxide (DMSO) (Cat No. D1435) were procured from Sigma-Aldrich (St. Louis, MO, USA), hematoxylin-eosin (Cat No. G1004), Masson staining kits (Cat No. G1006) and RPMI 1640 (Cat No. G4511) medium were provided by Servicebio (Wuhan, China). TRIzol Reagent (Cat No.15596018CN) was procured from Invitrogen (Waltham, MA, USA), the HiScript^® II Q RT SuperMix kit (Cat No. Q441-02) and the Power SYBR Green PCR Master Mix (Cat No. Q141-02) were obtained from Vazyme (Nanjing, China), dihydroethidium (Cat No. S0063) was procured from Beyotime (Shanghai, China), DMEM high-glucose medium (Cat No. BC-M-005) was provided by Bio-Channel (Nanjing, China). Antibodies against p-JNK (Cat No. a4668S) and p-p38 (Cat No. 4511S) were procured from Cell Signaling Technology (Danvers, MA, USA). Antibodies against HO-1 (Cat No. 10701-1-AP), c-Jun N-terminal kinase (JNK) (Cat No. 17572-1-AP), Nrf2 (Cat No. 16396-1-AP), NQO-1 (Cat No. 11451-1-AP), SOD2 (Cat No. 24127-1-AP), Gapdh (Cat No. 60004-1-Ig) and β-actin (Cat No. 66009-1-Ig) were obtained from Proteintech (Wuhan, China). CD68 (Cat No. ab283654), CD45 (Cat No. ab40763) and Ly6G (Cat No. ab238132) antibody were provided by Abcam (Cambridge, MA, USA).

Propolis (Henan Bei Yuan Bee products Co., Ltd.), Chitosan (Shandong Aokang Biotechnology Co., Ltd.), Polycaprolactone (Shanghai Yuan Ye Biotechnology Co., Ltd.), Polyvinyl alcohol (Shanghai Aladdin Biochemistry Technology Co., Ltd.), Dichloromethane, N, N-Dimethylformamide and Acetic acid (Yantai Far East Fine Chemical Co., Ltd.), Trypsin- EDTA digestive fluid, Streptomycin mixture and Heparin sodium (Beijing So Lar Bio-Technology Co., Ltd.), 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Shanghai Aladdin Biotech Co.), Commercial membranes (CM): Collagen sponge for hemostasis (Beijing All Gens Medical Instrument Co., Ltd.), Pentobarbital sodium salt (Merck & Co., Inc.), CaCl₂ (Tianjin Fengchuan Chemical Reagent Heparin sodium Co., Ltd.), Hematoxylin-eosin, IL-1β, IL-6, TNF-α, Antibodies vimentin, FITC-conjugated goat antirabbit IgG and FITC-conjugated goat anti-mouse IgG were obtained from Service bio, China.

The paraffin sections of rat brain tissues (5 μm) were deparaffinized by xylene and ethanol (2 × xylene, 100% ethanol, 100% ethanol, 95% ethanol, 75% ethanol, each 5 min), and stained with hematoxylin-eosin (Servicebio, China) method (hematoxylin 4 min, eosin 20 s). Then, sections were dehydrated in ethanol and xylene and sealed with synthetic resin.

Mice were sacrificed by cervical dislocation after anesthesia with tilotamine (0.09 mg/g), zolazepam (0.09 mg/g), and 0.01% xylazine (0.04 mL/g); and then their heart received a KCL injection in order to stop the heart in diastole. Heart from each group was excised and washed in cold PBS, fixed overnight in 10% formalin, and finally embedded in paraffin blocks. The slices were stained with hematoxylin-eosin and Sirius red (Servicebio; China) to evaluate cardiac remodeling. Cell area and interstitial fibrosis were quantified by Image J (NIH; United States).

The ankle joint longitudinal Sects. (5–6 μm) were baked in the 60 °C ovens for 1 h, deparaffinized in xylene, hydrated by ethanol, then stained with hematoxylin–eosin (Servicebio, China) and safranin O-fast green (Servicebio, China). Pathological changes were observed based on inflammatory cell infiltration, synovial hyperplasia, angiogenesis, and joint damage by a light microscope.