Alexa fluor anti rabbit antibody

In short, the circular glass slices were placed in six-well plates; transfected cell suspensions were added and then cultured in an incubator for 6 h in 5% CO₂ at 37°C to make cell climbing pieces. Subsequently, the climbing pieces were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and washed with PBS for three times. Cells were then permeabilized (PBS + Triton 0.1%) and blocked in 1% BSA for 1 h at room temperature. The primary antibody was diluted in the blocking buffer to incubate cells overnight at 4°C. Alexa Fluor anti-rabbit antibody (Invitrogen, Lot. A-11034) with green fluorescence was used as a secondary antibody and incubated at room temperature avoiding light for 1 h. After being stained with DAPI avoiding light at room temperature for 5 min, the slice was added with an appropriate amount of antifluorescence quencher and observed under a fluorescence microscope.

For endogenous LC3-II puncta staining, cells were washed in PBS and then fixed with 4% paraformaldehyde for 15 minutes at room temperature. Fixed cells were washed with PBS, permeabilized in 100% methanol for 10 minutes at –20°C, washed in PBS, and blocked in blocking buffer for 1 hour at room temperature. Cells subsequently were incubated with anti-LC3 antibody overnight at 4°C. After 3 TBST washes, cells were incubated with Alexa Fluor–anti-rabbit antibody (Invitrogen) for 2 hours at room temperature and then washed 3 times in TBST. Coverslips were mounted on slides using Vectashield anti-fade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen). Cells were observed under fluorescence microscope.
For ectopic eGFP-LC3 puncta, eGFP-LC3 plasmid was transfected into HepG2 cells with Lipofectamine 2000 Transfection Reagent (Invitrogen, Madison, WI, USA). Cells were observed under fluorescence microscope.
For autophagic flux analysis, tandem RFP/GFP-tagged LC3 plasmid was transfected into HepG2 cells with Lipofectamine 2000 Transfection Reagent (Invitrogen, Madison, WI, USA). Cells were visualized using LSM710 Carl Zeiss confocal microscope.