A1r a1

Following reperfusion, about 0.25 g liver samples were fixed with 10% buffered formalin for 24 h at room temperature (pH=7.2; cat. no. G2161; Beijing Solarbio Science & Technology, Co., Ltd.), then put into 75, 80, 85, 90, 95 and 100% alcohol for 1 h respectively, so as to fully dehydrate the tissues. Following dehydration, the tissues were soaked in 50% alcohol + 50% xylene solution for 30 min, and then soaked in 100% xylene until they became transparent. The transparent samples were soaked in liquid paraffin at 60°C twice (1 h each) until the paraffin had completely infiltrated. The samples after paraffin immersion were embedded in paraffin wax and cut into 4-µm sections for histological analysis via hematoxylin-eosin staining (H&E; hematoxylin staining for 5–15 min and eosin staining for 1–3 min; all performed at room temperature). Sections were analyzed under a confocal microscope (magnification, ×200; Nikon A1R/A1; Nikon Corporation) and images were obtained. A total of 6 fields of view per section were randomly selected for the assessment of liver damage. Numerical assessment of liver damage was conducted according to the histological criteria for assessment of liver damage.

At the end of the treatment period, all mice were euthanized with an overdose of sodium pentobarbital (200 mg/kg, i.p.). Animal death was verified by observation of cardiac arrest and pupil enlargement for 1 min. The humane endpoints established in this study were as follows: Impaired ambulation that prevented animals from reaching food or water; excessive weight loss and extreme emaciation; lack of physical or mental alertness; difficult labored breathing; and prolonged inability to remain upright. Animals were observed a minimum of twice daily, with more frequent observations immediately after dosing and when increased morbidity or mortality was expected (17 (link)). The heart was then removed and fixed in 10% formalin for 24–48 h at 4°C. Formalin-fixed heart tissues were then embedded in paraffin and sections were cut (~4 µm thick). Cardiomyocyte apoptosis was detected using a one-step TUNEL Apoptosis assay kit at 37°C for 1 h (Roche Diagnostics GmbH), according to the manufacturer's protocol, followed by DAPI staining (10 µg/ml; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 min. Anti-fade mounting medium was then added (cat. no. P0126; Beyotime Institute of Biotechnology) to each slide. Images were obtained from five fields per slide using confocal microscopy (magnification ×400; NIKON A1R/A1; Nikon Corporation).

GBC-SD cells were seeded into 24-well plates with carry sheet glass and treated with melatonin (2 mM) for 6 h. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were re-washed 3 times with PBS and YE Dye phalloidin conjugates (US Everbright) staining was performed according to the manufacturer's instructions. DAPI was used for nuclear staining at room temperature for 5 min. The images were acquired using a laser scanning confocal microscope (Nikon A1R/A1; Nikon Corporation) (magnification ×400).