Anti-LC3 pAb

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Stain with 10 µg/ml of antibody in blocking buffer for 1 h.

Protocol tips
- Stain with 10 µg/ml of antibody in blocking buffer for 1 h.

Publication protocol

Cells were plated on glass coverslips in 6-well tissue culture plates. After treatment as described in Mammalian cell culture and transfection, the cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min, and permeabilized in 0.1% saponin for 20 min. Fixed cells were blocked with 0.1% saponin and 10% FBS in PBS for 30 min, stained with 10 µg/ml of antibody in blocking buffer for 1 h, and washed with PBS three times every 5 min. Cells were then stained with secondary antibody (Alexa Fluor 488 Dye; Life Technologies) in PBS for 1 h and washed with PBS three times. Slides were mounted on glass coverslips using Fluoromount-G and imaged. Images were acquired using a confocal microscope (FV-1000; Olympus) that was equipped with Uplansapo 60×/1.35 oil and WHN 20×/22. Imaging medium was IMMERSIN OIL TYPE-E (Olympus). Image acquisition and processing were performed using FV10-ASW 3.1 software (Applied Precision). The mean environmental temperature during experimental acquisitions was 22°C. Images were copied to Illustrator (Adobe) and cut for the final figures.

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Manufacturer protocol

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