LC3B (D11) XP® Rabbit mAb

Protocol tips

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	Dilute primary Ab at 1:200 and incubate overnight at 4 °C

Protocol tips
Dilute primary Ab at 1:200 and incubate overnight at 4 °C

Publication protocol

HeLa cells and MEFs were seeded on coverslips in 12-well plates. After treatments, cells were fixed in 4% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilised with 0.5% Triton X-100 for 5 min at room temperature (or in methanol at −20 °C for LC3 staining). Coverslips were blocked for one hour in 5% normal goat or rabbit serum in PBS 0.05% Tween-20 (for LC3 staining, the Tween-20 was omitted from all steps). Cells were incubated with primary antibodies overnight at 4 °C. Primary antibodies used in this study include guinea pig α-p62 (Progen, 1:200), mouse α-p62 (BD Bioscience, 1:200), goat α-Ubiquitin (Santa Cruz Biotechnology #sc-34870, 1:200), rabbit α-LC3 (CST #3868, 1:200). Cells were washed three times and incubated with the appropriate secondary antibodies for 1 h at room temperature (Life Technologies, 1:5000). Cells were washed and nuclear DNA was stained by incubation with TO-PRO-3 iodide (Life Technologies, 1:3000) for 10 min at room temperature. Coverslips were mounted on slides with Prolong Gold Antifade (Life Technologies) and imaged with an LSM 510 META Confocal Microscope (Zeiss) using a 63× Plan-Apo/1.4 NA Oil objective. Images were analysed using ImageJ software (NIH).

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3B (D11) XP® Rabbit mAb below.

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