Alexa fluor 488 goat anti rabbit igg h l secondary antibody green

Kyse30 and Kyse450 cells were plated on a glass‐bottom cell culture dish and treated with 0.1% DMSO or PR‐619 (8 µmol/L) for 24 hours. Cells were fixed with methanol at −20°C for 30 minutes, blocked by 5% BSA and then incubated with LC3 primary antibody (1:200, overnight at 4℃) (Cell Signaling Technology, Inc, Boston, MA) and Alexa Fluor^® 488 Goat Anti‐Rabbit IgG (H + L) secondary antibody (green) (1:500, 2 hours at room temperature) (Beyotime, China), respectively. The nuclei were stained by DAPI (blue) (5 μg/mL, 20 minutes at room temperature) (Beyotime, China). Images were captured using fluorescence microscopy (magnification: 200×; Nikon, Nikon Inc, Tokyo, Japan).

HCCLM3 and SMMC-7721 cells were placed in a glass-bottom cell culture dish and treated with 0.1% DMSO or ML-323 (80 μmol/L) for 24 h. The cells were fixed with anhydrous methanol at −20 °C for 25–30 min, blocked with 5% bovine serum albumin, and then with LC3B primary antibody (1:300, overnight at 4 °C; Cell Signaling Technology) and AlexaFluor488® goat anti-rabbit IgG (H+L) secondary antibody (green) (1:500, 2 h at 25 °C in the dark; Beyotime) were incubated separately. The nuclei were stained with DAPI blue (5 μg/ml, 20 min at 25 °C, in the dark; Beyotime). A fluorescence microscope (magnification:200×; OLYMPUS, OLYMPUS Corporation, Japan) was used.