LC3B Antibody Kit for Autophagy

Protocol tips

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	primary antibody dilution 1:500 and incubate for 2h

Protocol tips
primary antibody dilution 1:500 and incubate for 2h

Publication protocol

Cells were washed with PBS, fixed with 2% formaldehyde in PBS for 20 min, washed with PBS, fixed and permeabilized with pre-chilled (−20°C) 70% ethanol for 20 min, washed with PBS, blocked with 8% bovine serum albumin (BSA) in PBS for 1 h, washed with PBS, incubated with the first antibody: anti-LC3-A/B (L10382, Invitrogen), 1/500 dilution; anti-γH2AX (NB100-384, Novus Biologicals), (1/500 dilution); anti HNE (393206, Calbiochem, San Diego, CA), (1∶500 dilution), in 1% BSA in PBS. After incubation for 2 h, slides were washed with PBS, incubated with the secondary antibody conjugated with Alexa Fluor 488 or 568 for 1 h at room temperature, washed with PBS, and mounted with Vectashield mounting medium with DAPI (4′, 6′-diamidino-2-phenylindole) to counterstain the DNA (Vector Laboratories, Burlingame, CA). Slides were examined using a laser scanning confocal microscope (Zeiss LSM510). Images were collected and processed using the Zeiss AIM software. Level of fluorescence intensity was realized with ImageJ software.

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