LC3B (D11) XP® Rabbit mAb

Protocol tips

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	Dilute primary Ab at 1:200

Protocol tips
Dilute primary Ab at 1:200

Publication protocol

A Nikon Eclipse E800 Fluorescence microscope was used. Cells were plated on coverslips one night before being transfected with indicated constructs in Opti-MEM using FuGENE HD reagent. 24 h after treatment, slides were fixed using 4% paraformaldehyde/PBS at room temperature for 15 min and washed twice with PBS. For immunofluorescent analysis, slides were then washed twice with PBS, permeabilized by using PBS containing 0.25% Triton X-100, and blocked with 2% BSA in PBST for 30 min, and the first antibodies were added following Abcam immunofluorescence protocol. The autophagic process was analyzed by counting percentage of cells showing accumulation of GFP (RFP) LC3 puncta and the average number of GFP (RFP) LC3 puncta per cell. For the calculation of percentage of cells showing LC3 accumulation, a total of 300 cells were calculated each time for three duplicates. For the calculation of average number of puncta per cell, a total number of 100 cells were calculated each time for three duplicates. The representative fluorescent images were collected using SPOT Advanced software.

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Manufacturer protocol

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