2.3　Immunofluorescence staining

MIN-6 cells were cultured in 12-well plate which placed round glass coverslips.After discarding the medium，cells were washed with phosphate buffered saline(PBS)three times and fixed with 4% paraformaldehyde for 10min，then washed with PBS three times.After blocking with PBS plus 10% FBS for 30min，cells were incubated with anti-LC3 primary antibody in 37℃for 1 hr，after washing with blocking PBS three times and incubated with secondary antibody(Alexa Fluor 488 Goat Anti-Rabbit A11034，Invitrogen Life technology；Shanghai，China)in 37℃for 1hr，then washed with PBS three times.Images were performed with confocal microscopy(Zeiss Laser Scan Microscopy 710，Germany).(https://www.daowen.com)