Anti-LC3B antibody produced in rabbit

Protocol tips

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	Dilute primary Ab at 1:1000 and Incubate primary antibody overnight at 4 °C

Protocol tips
Dilute primary Ab at 1:1000 and Incubate primary antibody overnight at 4 °C

Publication protocol

HeLa cells were cultured on cover slips and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature. After washing three times with PBS, the cells were permeabilized with ice-cold methanol for 2.5 min. After three washes with PBS, the cells were incubated with blocking solution (5% BSA in PBS) for 1 h and then with primary antibody overnight at 4 °C. The next day, the cells were washed five times for 5 min each time with PBS and then incubated with secondary antibody for 1–2 h. The cells were washed four times with PBS for 5 min each time, and DAPI stained for 15 min. After three washes with PBS, the coverslips were mounted on slides using Fluoro-GEL (Electron Microscopy Sciences). Confocal images were taken by laser scanning confocal microscope 510 Meta (Zeiss) and analyzed using Zeiss LSM Image Browser (ver. 4.2.0.121). To investigate the effect of XIE62-1004 and XIE2008 compounds on p62 puncta formation, cells were incubated with different concentrations of XIE compounds (0, 1, 2.5, 5, and 10 μM) for 12 h or incubated with 10 µM XIE compound for varying time periods (0, 1, 3, 6, and 12 h). To analyze the physical association of p62 puncta with autophagic vacuoles containing LC3, HeLa cells were left untreated or treated with 10 µM XIE62-1004 or XIE2008 compound for 16 h followed by immunostaining with anti-p62 and LC3 antibodies.

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