Plan apochromat 63 1.4 na oil dic objective

For the detection of LAS1L by immunofluorescence, cells grown on cover glass were fixed with 4% paraformaldehyde at room temperature for 15 min. Cells were then permeabilized at room temperature with 0.1% Triton for 10 min. All samples were blocked with 1% BSA for 30 min, washed with PBS, and incubated with an anti-LAS1L antibody (Cat#: AV34629, Sigma) for 1 h. Cells were washed with PBS containing 0.05% Tween-20 and incubated with secondary antibody for 1 h (Alexa-Fluor 488 goat anti-rabbit antibody, Cat#: A11001, Invitrogen). Cells were washed again with PBS containing 0.05% Tween-20, stained with DAPI (Molecular Probes), and mounted on slides with Vectashield (Vector Labs). To visualize LC3 localization to autophagosomes, HCT116 cells were transfected with a plasmid expressing pEGFP-LC3 (a gift from Toren Finkel (Addgene plasmid #24920; Lee et al., 2008 (link)) and pools of stable clones were established after selection with G418. Fluorescence microscopy was performed on a Zeiss Axioskop 40 fluorescence microscope with a Plan-APOCHROMAT 63×/1.4 NA oil DIC objective. Images were acquired with an Axiocam MRm camera using the Axiovision Release 4.6 software with a capturing resolution of 1388 × 1040 pixels. All microscopy was performed at room temperature, and all images were prepared in Adobe Photoshop and Adobe Illustrator.