Tannic acid acs reagent

Folin-Ciocalteu reagent, HPLC grade methanol, tannic acid ACS reagent, Quillaja saponin, 3,5-dinitrosalicilic acid (98 %), aluminum trichloride, sulfuric acid (18 M), glucose (99.5 %), stigmasterol (95 %), hydroquinone (99 %), ursolic acid (98.5 %), digitoxin (92 %) and bromocresol green (95 %) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Quercetin dihydrate (98 %) and dragendorff’s reagent were obtained from Aldrich (Milwaukee, WI, USA). Ferric Chloride (FeCl₃•6H₂O) (97.0-102.0 %) was obtained from Spectrum Chemical and Lab Products (Gardena, California, USA) and potassium ferrocyanide (K₄Fe(CN)₆•3H₂O), HCl (12 M), sodium hydrogen phosphate, ethyl acetate (EtOac), acetic anhydride and dichloromethane were obtained from Thermo Fisher Scientific (New Jersey, USA). Nicotine (99 %) was obtained from VWR (New Jersey, USA). All chemicals were used without further purification.

EVs: MSC‐EVs were fixed using 500 µL of 4% paraformaldehyde. Then, the EVs were resuspended in 30 µL of PBS and then 10 µL of each sample was placed on formvar/carbon‐coated grids, dried at room temperature, and then fixed with 2% glutaraldehyde for 10 min. Next, 1% tannic acid ACS reagent (Sigma‐Aldrich, UK) and TAAB EM Heavy Metal Stain 336 (TAAB, Laboratory Equipment Ltd., UK) were used for staining. Finally, MSC‐EVs were visualized using a TEM microscope (HT7800, HITACHI, Japan).
Cells: 1 × 10⁶ cells were fixed in 2.5% glutaraldehyde (pH 7.4) at 4 °C for 24 h. Cells were precipitated by embedding in 1% agarose and then fixed in 1% osmium acid for 2 h and dehydrated in a gradient series of acetone and ethanol. Next, ultra‐thin sections of 60 nm thickness were prepared using an ultra‐thin sectioning machine (UC7, Leica, America). These sections were then stained with 2% dioxygen acetate and 2.6% lead citrate. Finally, the sections were observed by TEM microscopy.