Transmission electron microscopy

After establishing I/R model, myocardium (1 mm³ ) were incubated in 2.5% glutaraldehyde for 4 h. Following fixation in 1% osmium tetroxide for 1 h, samples were dehydrated with various concentration of alcohol. Finally, samples were embedded and stained. Transmission electron microscopy (FEI, Hillsboro, OR) was used to analyze the samples.

Liver tissues from animal models were fixed in 10% buffered formalin for 8 h, followed by transfer to 70% ethanol, then embedded by paraffin. Sectioned liver tissues (5 μm) were stained by H&E according to the manufacturer’s protocol. For IHC staining, we performed the experiment as described^{48 (link),49 (link)}. The primary antibodies to NRF2 (1:200 working dilution), GCLC (1:100 working dilution), GXP4 (1:100 working dilution), and 4-HNE (Abcam, ab46545; 1: 100 working dilution) were commercially obtained. Negative control slides were performed without primary antibody. Control slides known to be positive for each antibody were incorporated. To quantify the IHC result of positive staining, the tissue areas of five ducts (173 mm²) in each sample were microscopically examined and analyzed by an experienced pathologist. Liver tissues (1 × 1 × 1 mm) were fixed by glutaraldehyde, and observed under transmission electron microscopy (FEI, Hillsboro, USA) at the Electron Microscopy Core Facility, Xiangya Hospital, Central South University. Images were captured using a charge-coupled device camera and analyzed using Motic Images Advanced software.

DOPA-capped NCP nanoparticles were prepared according to our previous report16 (link). NCP@pyrolipid was prepared by adding a THF solution (80 μl) of 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, pyrolipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine polyethylene glycol 2000 (2:1:0.8:1 in molar ratio) and DOPA-coated NCP to 500 μl of 30% (v/v) ethanol/water at 60 °C. The mixture was stirred at 1,700 r.p.m. for 1 min. THF and ethanol were completely evaporated and the NCP@pyrolipid solution was allowed to cool to room temperature (r.t.). NCP@pyrolipid was centrifuged at 19,650 g. for 30 min. The supernatant was then removed and the particles resuspended in PBS. ICP-MS (Agilent 7700X, Agilent Technologies, USA) was used to analyse the Pt concentration of NCP to calculate oxaliplatin loading. The particle size and zeta potential of NCP@pyrolipid in PBS were determined by Zetasizer (Nano ZS, Malvern, UK). Transmission electron microscopy (Tecnai Spirit, FEI, USA) was used to observe the morphology of NCP@pyrolipid.