Osmic acid

Cells were collected by centrifugation after trypsinization and immediately fixed in 2.5% glutaraldehyde (Sigma-Aldrich) for 24 h and then in 1% osmic acid (Sigma-Aldrich) for 1–2 h, dehydrated with acetone (Sinopharm Chemical Reagent, Shanghai, China), and embedded in araldite CY 212 (TAAB, Aldermaston, UK). The ultrathin sections were stained with alcoholic uranyl acetate (Polysciences, Warrington, USA) and alkaline lead citrate (Sigma-Aldrich), washed gently with distilled water, and observed with a JEM 1230 transmission electron microscope (JEOL Ltd, Tokyo, Japan).

The OC-1 cells were fixed in 2.5% glutaraldehyde (Sigma-Aldrich) for 24 h and 1% osmic acid (Sigma-Aldrich) for 1–2 h, dehydrated with acetone (Sinopharm Chemical Reagent), and embedded with Araldite CY212 (TAAB). Ultrathin sections were stained with alcohol uranyl acetate (Polysciences) and alkaline lead citrate (Sigma-Aldrich). The sections were gently washed with distilled water and observed under a JEM 1230 transmission electron microscope (JEOL Ltd.).

Graphite powder (G), potassium bromide (KBr), hydrogen peroxide (H₂O₂), chitosan 448869, acetic acid, Hoechst 333,242 (HS), propidium iodide (PI), glutaraldehyde (50%), osmic acid (4%), Nafion solution, 2′,7′-dichlorodihydroflourescein diacetate (DCFA-DA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Potassium permanganate (KMnO₄) and trisodium citrate (Na₃C₆H₅O₇) were purchased from Nihon Shiyaku Industries Ltd., Osaka, Japan. Sulfuric acid (H₂SO₄, 95–98%) solution was purchased from Scharlab S.L., Barcelona, Spain. Silver nitrate (AgNO₃) was purchased from Mallinckrodt Baker Inc., Paris, France, and hydrochloric acid (HCl) was purchased from Showa Chemical Co., Ltd., Honshu, Japan.

Cells were detached from the plates using 0.25% trypsin trypsin-EDTA (Gibco Life Technologies) and fixed with 2% paraformaldehyde/2% glutaraldehyde (Sigma-Aldrich) in 0.2 M sodium cacodylate buffer (pH 7.4; Sigma-Aldrich). Cell pellets were post-fixed with 1% (v/v) osmic acid (Sigma-Aldrich) in sodium cacodylate buffer and were stained with 1% uranyl acetate (Amresco, Solon, OH, USA). Following dehydration, the pellets were embedded in Durcupan (Sigma-Aldrich). Ultrathin sections (50 nm) were prepared using an Ultrotome Ultracut S (Leica Microsystems, Wetzlar, Germany) and images were captured with a JEM-1230 transmission electron microscope (JEOL, Ltd., Tokyo, Japan).

The left lungs of the mice were fixed in 2.5% glutaraldehyde (Sigma, St. Louis, MO, USA) in PBS for 2 h. Then, the lungs were rinsed three times with PBS and exposed to 1% osmic acid (Sigma) for 2 h, followed by an additional three washes with PBS. Then, the lungs were dehydrated using an ascending alcohol series and infiltrated with Epon 812 overnight. The Epon 812-infiltrated tissues were placed in molds and polymerized at 80°C for 48 h. Ultrathin sections of the resulting blocks were cut to 60 nm using a Leica microtome and settled onto 400-mesh copper grids. The samples were stained for 15 min with 3% uranyl acetate and for 10 min in 1% lead citrate, followed by three washes with double-distilled water to take excess stain away. The samples were observed on a TEM (Hitachi H-7650, Japan).

Samples were fixed in 1% osmic acid (Sigma, USA) for 30 min at room temperature and dehydrated by an ascending series of ethanol (from 50 to 100%). After air-drying overnight, samples were coated with gold. SEM (VegaII XMU instrument Tescan, Czech-Republic) was used to observe the morphology of ECSs.