H e staining kit

Livers were fixed into 4% parafomaldehyde. Then the tissues were embedded in paraffin and sectioned for 5 μm thick. Hematoxylinensin (H&E) staining was used for liver pathological evaluation. H&E staining kits were obtained from Jiancheng Bioengineering Institute (Nanjing, China). All kits were used according to the corresponding manufacturers’ instructions Liver steatosis score was numerically recorded following the method of Qayyum et al. (17 (link)).

Four mice from each group were killed on days 3, 7, 11, and 15. Wounds with the surrounding skin were excised. The specimen was harvested with a 2-mm border of unwounded skin tissue, and then fixed in 10% formalin and later embedded in paraffin. The tissue blocks were then cut into 5 μm sections, transferred to glass slides for hematoxylin and eosin (H&E) staining (HE staining kit, Nanjing Jiancheng Bioengineering Institute, Nanjing, China, batch No. 20120926). Morphological alterations in the skin tissues were examined by light microscopy and documented by photographs.

Twelve weeks post-transplantation. specimens were dehydrated and embedded in paraffin according to standard protocols. Semi-thin sections were stained with HE staining kit (JianCheng Biotech, China) or toluidine blue (JianCheng Biotech, China). Images were photographed by an inverted phase contrast microscope.

SCSP was synthesized by Wuxi MimoTopes Biotechnology (Wuxi, China) [13 (link)]. Cyclophosphamide was supplied by Hengrui Medicine (Lianyungang, China). BUN, CRE, GSH-Px, MDA, SOD, CAT determining kits and the H&E staining kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kits and PAS stain kit were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). The primary antibodies against β-actin, NF-κB p50, NF-κB p65, IKKα, IKKβ, and IκBα were supplied by Beyotime Biotechnology (Shanghai, China). The monoclonal antibodies against Bcl-2, Bax, caspase 3, and caspase 9 were provided by Cell Signaling Technology Inc. (Beverly, MA, USA). The primary antibodies against TNF-α were supplied by Proteintech Group, Inc. (Wuhan, China). All other reagents were analytically pure.

Fixed liver tissue was dehydrated and embedded using a tissue processor (Leica ASP300) and paraffin embedding station (Leica EG1160). Then, the sections were stained using an HE staining kit (lot number 20180530, Nanjing Jiancheng Bioengineering) and a Sirius red staining kit (lot number 20180528, Nanjing Jiancheng Bioengineering).

TAA (purity ≥ 99.0%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 20 (R)-G-Rg3 (purity ≥ 98.0%, HPLC) was obtained and qualified as described by our previous study^{26 (link)}. mTOR inhibitor Rapamycin (Ra) and PI3K inhibitor LY294002 were obtained from Med Chem Express Biotech Co. Ltd. (New Jersey, USA). Catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), H&E staining kit, and Masson staining kit were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kit for TGF-β1 was purchased from R&D systems (Minneapolis, MN, USA). Autophagy-related antibodies of LC3 a/b (12741 S), ATG3 (3415 S), ATG5 (12994 S), ATG7 (8558 S), ATG12 (4180 S), ATG16L (8089 S), Beclin-1 (3495), mTOR (2972 S), p-mTOR (2971 S), p-ULK1 (14202), Akt (9272 S), p-Akt (13038), PI3K (4292 S), p-PI3K (422S8), and anti-HRP were from Cell Signaling Technology (Massachusetts, USA). p62 (18420-1-AP), α-SMA (23660-1-AP), TGF-β1 (21898-1-AP), β-actin (60008-1-Ig), and GAPDH (60004-1-Ig) were from Proteintech (Chicago, USA). All other reagents and chemicals, unless indicated, were obtained from Beijing Chemical Factory (Beijing, China).