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Immunofluorescence staining and co-localization analysis

YC Yiran Chen
XX Xiaoling Xie
CW Chunsheng Wang
YH Yuxing Hu
HZ Honghao Zhang
LZ Lenghe Zhang
ST Sanfang Tu
YH Yanjie He
YL Yuhua Li
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Cells were suspended in DMEM with 10% FBS and seeded in 24-well plate with coverslips. After overnight culture, cells were treated with corresponding drugs (WZ4003, SBI-0206965, chloroquine, MRT68921, or combinations) for different time. After treatment, cells were fixed by paraformaldehyde and treated with 5% TritonX-100 for 20 mins. After confined with 2% BSA, cells were incubated with fluorescent antibodies (ULK1: Abcam, #ab240916, 1:50; LC3B: CST, 1:50) overnight. Then, cells were cultured with secondary antibody (Alexa Fluor 488-Goat Anti-Rabbit IgG(H + L), Proteintech, 1:100) for 2 h in dark. After that, cells were treated with mitotracker and DAPI in turn. Coverslips were upside-down on slides with a sealing agent and observed under immunofluorescence microscope or laser scanning confocal microscope.

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