Masson staining kit

Harvested the whole hind limbs at 14 and 21 days after post-repair [24 (link)] fixed them in 10% neutral buffered formalin for 48 h and washed in phosphate-buffered saline (PBS), then decalcified in 14% EDTA (pH 7.2) for 14 days at room temperature. The decalcified tissues were dehydrated with gradient ethanol, subjected to xylene transparency, dipped in wax, embedded in paraffin, and cut into 5 μm slices. Finally, H&E (Beyotime, China) and Masson staining kit (Beyotime) were used for sections staining by the manufacturer's instructions. Finally, they were dried and coated in neutral resin, followed by light microscopy. ImageJ image analysis software was used for analyzing the area of fibrosis (%) in samples.

Lung tissues were fixed with 4% paraformaldehyde for 24 h and underwent dehydration by alcohol of different concentration. Tissues were embedded into paraffin and placed at room temperature for 24 h, then cut into 5 μm sections. Haematoxylin-eosin (HE) staining kit (cat. no. C0109; Beyotime) and Masson staining kit (cat. no. C0215; Beyotime) were used to determine the degree of alveolitis and fibrosis withSzapiel’s method (Szapiel et al., 1979 (link)). Images were captured under a microscope (BA210T; Motic). For IHC, the sections received antigen retrieval in citrate buffer at 95°C for 15 min, then blocked with 0.5% BSA-PBS containing 10% goat serum for 1h, and finally incubated with anti-Fibronectin (1:100; 66042-1-IG), anti-α-SMA (1:200; 55135-1-AP), or anti-Sirt2 (1:100; 19655-1-AP) antibody overnight. The density of positive areas was measured using Image-Pro Plus 6.0 software.

AFB1 was purchased from Macklin.D ihydromyricetin was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China) BCA Protein Concentration Assay Kit, CAT Kit, MDA Kit, SOD Kit, ALT Kit, GPX Kit, GST Kit and AST Kit were purchased from Solarbio Biotechnology Co., Ltd. (Beijing, China). Electronic balance was purchased from Shanghai Jingtian Electronic Instrument Co., Ltd.(Shanghai, China).model FA2104A. The 1- -16K cryocentrifuge was purchased from Shanghai Trading Co., Ltd. (Shanghai, China). model D37520. H&E staining kit, Masson staining kit and TUNEL apoptosis assay kit. The 16K freezing centrifuge was purchased from Shanghai Trading Co., Ltd. (Shanghai, China). model D37520. H&E staining kit, Masson staining kit, and TUNEL apoptosis detection kit were purchased from Beyotime Biotechnology Co. (Shanghai, China). Rabbit anti-mouse, Nrf2, Keap1, Bax, Bcl-2, HO-1, Caspase-3, Cleaved-Caspase-3, and GAPDH monoclonal antibodies were purchased from Abcam (Cambridge, UK). All other chemical reagents used in the experiments were of analytical grade provided by Beijing Chemical Factory (Beijing, China).