Paraformaldehyde solution

The order of the procedures for histopathology and immunohistochemistry was as follows: 1) tissue preparation, 2) fixation, 3) dehydration, 4) embedding, and 5) staining.
The liver and kidneys were obtained from the mice and fixed overnight at 25°C in a 4% paraformaldehyde solution (Biosesang, Seoul, Korea). The fixed liver was cut through the caudate and left lateral lobes. The fixed kidneys were transected. The dissected organs underwent tissue processing using a tissue processor (TP1020^®; Leica Biosystems, Lincolnshire, IL, USA). The dissected organs were dehydrated through a series of graded ethanol baths to displace water and then infiltrated with wax. The organs were embedded in paraffin using an embedding center (EG1150^®; Leica Biosystems). Tissue sections were cut to 4-μm thickness using a microtome (Leica Biosystems) and mounted on poly-l-lysine coated microscopic slides (Mutokagaku, Tokyo, Japan).

The exosomes extracted from heart tissues were first prepared for TEM analysis by fixing them with a 0.1% paraformaldehyde solution (Biosesang, Seongnam, Republic of Korea) for 30 min. This step is critical in preserving the structural integrity of the exosomes during the subsequent analysis steps. A 10 μL aliquot of each exosome sample was placed on a piece of Parafilm. A formvar/carbon-supported copper grid (200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA) was then floated on top of each sample drop for 7 min [27 (link)]. This method allowed the exosomes to adhere to the grid while being sufficiently supported for detailed TEM examination. The grid with adhered exosomes underwent a washing procedure, alternating with three drops of ultrapure water, each wash lasting for a 2 min duration. This step ensured the removal of any residual fixing agent. Subsequently, the grid was stained with a 2% solution of phosphotungstic acid (pH 7.0; Sigma-Aldrich, St. Louis, MO, USA) for 30 s, which provided the contrast necessary to visualize the exosomes under TEM. After staining, the grid was air-dried overnight in a dark environment to prevent any light-induced alterations. Once dried, the exosomes were ready for visualization. The prepared samples were examined under a transmission electron microscope (JEM-F200; JEOL, Tokyo, Japan).

Co-cultured tissues in the device were fixed with 4% (w/v) paraformaldehyde solution (Biosesang, Seongnam, Korea) in PBS (Welgene, Gyeongsan, Korea) for 20 min, followed by permeabilization with a 20 min immersion in 0.15% Triton X-100 (Sigma, St. Louis, MO, USA). The samples were then treated with 3% BSA (Sigma, USA) for 1 h. The cells were incubated with antibodies purchased from Abcam. The primary antibodies were anti-cytokeratin 8 and anti-α-SMA, left for 2 days at room temperature (RT). Alexa Fluor 647 conjugated donkey anti-rabbit IgG (H + L) was used as the secondary antibody. DNA labeling was performed with a 1:250 dilution of Hoechst 33342 (Thermo Scientific, Rockford, IL, USA) for 3 h at RT. Images were collected using a Zeiss LSM 710 confocal laser microscope. The mean fluorescence intensity (MFI) was measured using ImageJ and a fluorescence-intensity analyzer.