LC3B Antibody #2775

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	Incubate the blots with primary antibody in 5% non-fat dry milk in PBS plus 0.1% Tween-20 overnight at 4°C

Protocol tips
Incubate the blots with primary antibody in 5% non-fat dry milk in PBS plus 0.1% Tween-20 overnight at 4°C

Publication protocol

Confocal microscopy
Autophagic flux was measured in the mCherry-LC3B stable cell line. Astrocytes expressing Cherry-LC3 were grown on a coverslip, treated with different conditions and fixed with 4% paraformaldehyde for 15 min in PBS. The coverslips were mounted with antifade (SlowFade; Life Technologies) and examined under a confocal microscope (TCS SP2; Leica). The results report the number of LC3 puncta per cell. For quantification of red (Cherry) mCherry-LC3 (td-tag-LC3) puncta, pictures were captured at 60× magnification on a confocal microscope, and the number of red puncta analysed as described above from (30–40) randomly selected cells per experiment and condition.

Western blotting assays
Cells for each different experimental protocol were lysed in RIPA (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris pH 8.0, and 2 mM MgCl2). Protease and phosphatase inhibitors (protease inhibitor cocktail plus 5 mM sodium fluoride, 0.5 mM sodium orthovanadate, 1 mM sodium molybdate, 50 mM 2-chloroacetamide, 2 mM 1,10-phenanthroline monohydrate, and 0,5 mM PMSF; Sigma-Aldrich) were added. Lysates were incubated at 4°C for 30 min. After centrifugation at 4°C for 10 min at 13,000 rpm to remove insoluble debris, the protein concentrations were determined using a Bradford assay with bovine serum albumin as the reference standard (Biorad). Equal amounts of protein (10 μg) were re-suspended in SDS-PAGE sample buffer. The samples were then separated on NuPAGE Bis-Tris gel (Life Technologies) and electroblotted onto nitrocellulose (Protran, Schleicher & Schuell) or PVDF (Millipore) membranes. Blots were incubated with primary antibody in 5% non-fat dry milk in PBS plus 0.1% Tween-20 overnight at 4°C. Detection was achieved using a horseradish peroxidase-conjugate secondary antibody (Jackson Immuno Research Laboratories) and visualised with ECL (GE Healthcare).

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