Exosome and Slide Immunofluorescence Staining

The samples (exosomes and slide) were previously fixed for 10 min in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Before staining the samples, they were washed 3 times on PBS and incubated for 1 h at room temperature in PBS solution plus 3% bovine serum albumin (BSA, Sigma-Aldrich). After subsequently washing the samples, they were incubated at 4 °C overnight with primary mouse antibodies anti-human CD 81, CD63, (Thermo Fisher Scientific, Waltham, MA, USA) and with fluorescent secondary goat antibodies anti-human (Alexa Fluor 555 dye, Thermo Fisher Scientific). Additional staining was also performed. Next, the exosomes were marked with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St. Louis, MO, USA) for 5 min at room temperature in a dark room and blocked with fetal bovine serum, according to the manufacturer’s instructions [49 (link)].