Tecnai g2 spirit

EV were fixed with 2% phosphotungstic acid at 4 °C, air-dried, observed under a TEM at 80 kV (FEI Tecnai G2 Spirit, Thermo Scientific, USA) and analyzed by western blot [30 (link)].

The HiPSC-ECs were cultured to 70% confluency, and the supernatant was collected and centrifuged at 2,000 g for 10 min at 4°C to remove the residual cells and debris followed by another centrifugation at 100,000 g for 1 h at 4°C. Afterward, the EV pellets were resuspended in 50–100 μl of phosphate-buffered saline (PBS). EVs were visualized with a transmission electron microscopy (FEI Tecnai G2 Spirit, ThermoFisher Scientific, Waltham, MA, USA) directly or an inverted fluorescence microscope (Olympus FV3000). The diameter distribution of EVs was determined by NanoSight (Malvern Instruments Inc., Malvern, UK). Some specific protein markers, such as CD9, CD63, and TSG101, were identified by Western blotting analysis.

Exosomes were dropped onto the copper grid and negatively stained with 2% phosphotungstic acid for 2 min. After air-drying, the samples were observed using a transmission electron microscope (FEI Tecnai G2 Spirit, Thermo Scientific, USA) at 80 kV.

Karnovsky fixed colonic samples were further processed by post-fixation at 4 °C with 1% osmium tetroxide in sodium cacodylate (0.1 M) containing potassium ferrocyanide (0.8%) and subsequently dehydrated with ethanol. In the final step, fixed colonic tissue was infiltrated, embedded and polymerized into Epon resin for at least 48 h at 60 °C. Ultrathin sections (70 nm thick) were cut using an ultramicrotome (Ultracut UC6, Leica Microsystems). For TEM imaging, saline (N = 4) and UP-exposed (N = 6) colonic samples were randomly selected and imaged. For human samples, control (N = 2) and NEC (N = 2) colonic samples were used. The ultrathin-cut sections were mounted on Formvar-coated copper grids (single slot grids) and subsequently stained with 2% uranyl acetate in 50% ethanol and lead citrate. The TEM-prepared colonic samples were imaged using a Tecnai G2 Spirit transmission electron microscope with a CCD Eagle camera (4 k × 4 k camera; Thermo Fisher Scientific).

For electron microscopy analysis, exosomes were dropped onto the copper grid and negatively stained by 2% phosphotungstic acid for 2 min. After air-drying for 15 min, the samples were observed under a transmission electron microscope (FEI Tecnai G2 Spirit, Thermo Scientific, USA) at 80 kV.

Cell or tissue samples were collected and fixed with 4% pre-cooled glutaraldehyde (Sigma-Aldrich Chemical, St. Louis, USA) overnight at 4 °C, followed by PBS washing on the next day. A series of treatments were subsequently applied, including 1% osmic acid fixing, water washing, 2% uranyl acetate staining, gradient concentration ethanol, and acetone dehydrating. Consequently, the dehydrated samples were embedded and sliced into 100 nm samples. The morphologies of mitochondria were observed by TEM (Tecnai G2 spirit, Thermo Fisher Scientific, Waltham, USA). Mitochondria with healthy morphology were counted and the area and perimeter of mitochondria were quantified by Image J software (six independent lung sections for each group).