Anti lc3 clone 2g6

Immunostaining for LC3 was performed with blocking and permeabilization buffer (0.3% Triton X-100, 2% bovine serum albumin) and anti-LC3 (clone 2G6, Nanotools #0260–100/LC3-2G6) at a dilution of 1:200 for 3 hr at room temperature. Samples were incubated with a secondary antibody conjugated to Alexa Fluor 647 fluorophore at a dilution of 1:1000 (ThermoFisher) and DAPI at a dilution of 1:1000 for one hour at room temperature. 59 images per well were obtained with the Opera Phenix confocal microscope at 40X magnification for colocalization or 63X for figures. Automated colocalization measurements were performed in macrophages infected with two Mtb bacilli.

All the reagents and chemicals were obtained from Sigma–Aldrich, unless otherwise indicated. Lipo-polysaccharide (LPS) (L2800), chloroquine diphosphate (CQ) (C6628) & Rapamycin (RAPA) was obtained from LC laboratories (R-5000). Sources of the antibodies were as follows: HSC70 (Thermo Scientific, PA5-27,337), anti-CD9 (Abcam, ab92726), Bcl2 (Cell Signaling, D17C4), nucleoporin P62 (BD Transduction Laboratories, 610,497), anti-GM130 (BD Transduction Laboratories, 610,822), anti-β-actin mAb (Sigma–Aldrich, A5441), anti-LC3 (clone 2G6) (NanoTools, 0260-100).

Unless otherwise indicated, all the reagents and chemical were obtained from Sigma–Aldrich. Proteinase K (PK; 03115879001) and Pefabloc inhibitor (11286700) were from Roche. Lipofectamine 2000 (Invitrogen, 1166807), Lipofectamine LTX transfection reagents (Invitrogen, 15338100), and On Target plus mouse Atg5 (11793) siRNA Smart Pool (GE Health Care, L-064838-00-0005) were acquired from the manufacturers indicated in parentheses.
Sources of the antibodies were as follows: anti-flotillin-1 (BD Transduction Laboratories, 610820), anti-Tsg101 (Santa Cruz Biotechnology, sc-7964), HSC70 (Thermo Scientific, PA5-27337), CD63 (Santa Cruz Biotechnology, SC-15363), Alix (Cell Signaling, 2171), anti-CD9 (Abcam, ab92726), Bcl2 (Cell Signaling, D17C4), Atg-5 (clone 7C6) (NanoTools, 0262-100), nucleoporin P62 (BD Transduction Laboratories, 610497), anti-GM130 (BD Transduction Laboratories, 610822), anti-β-actin mAb (Sigma–Aldrich, A5441), anti-LC3 (clone 2G6) (NanoTools, 0260-100), anti-PrP mAb 4H11 has been previously described (52 (link)). Peroxidase-conjugated Igs were from Jackson Immunoresearch Lab (goat anti-mouse HRP, 115-035-003; and goat anti-rabbit HRP, 111-035-45).