Alexa fluor 488 labeled goat anti rabbit

Cells were treated with various drug combinations as indicated for 7 days, whereas 50 μM chloroquine was added for the final 16 hours. Live cells were stained with 50 nM LysoTracker Red DND-99 (LY528, Invitrogen) in prewarmed medium for 15 minutes at 37°C. Cells were plated, fixed, permeabilized, and blocked using the same method as the E-cadherin (CDH1) and vimentin (VIM) staining. Cells were then incubated in primary Ab LC3 A/B (DU4C) (12741, Cell Signaling Technology) at one-to-100 ratio overnight at 4°C, carefully rinsed three times with PBS-T, and incubated with secondary antibody Alexa Fluor-488 labeled goat anti-rabbit (A11008, Invitrogen, 1:200) for 1 hour at room temperature; it was then subsequently washed four times with PBS-T before mounting with ProLong Diamond Antifade Mountant with DAPI (P36962, Molecular Probes). This protocol was adapted from Kang et al.^{34 (link)} Images were obtained on a Leica TCS SP8 confocal microscope using 100× objective lens magnification (HC PL Apo STED white, oil, NA = 1.4, WD = 0.13 mm). The co-localization intensity spatial profile was obtained by drawing a one pixel-wide square across the images, and the same area was selected for the green and red channels for each image. The plot profile function in ImageJ was then used to find the intensity values that were plotted in GraphPad Prism.

HepaRG cells were rinsed in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 20 minutes, and quenched with 10 mM glycine in PBS. Cells were permeabilized for 5 minutes in 0.2% Triton-X100/PBS, followed by a 30-minute blocking step with 1% bovine serum albumin (BSA) in PBS. Cells were then incubated for overnight at 4°C with the following primary antibodies: rabbit anti-ZIP14 (Sigma 1:200), mouse anti-MRP2 (Abcam, 1:200), mouse anti-Lamp1 (Abcam, 1:100), mouse anti-beta1 NaK-ATPase (Abcam, 1:200, followed by a 45 minutes incubation with secondary antibodies AlexaFluor568-labeled goat anti-rabbit (Molecular Probes, 1:500), AlexaFluor568-labeled goat anti-mouse (Molecular Probes, 1:500), AlexaFluor488-labeled goat anti-rabbit (Invitrogen, 1:500), or AlexaFluor488-labeled goat anti-mouse (Invitrogen, 1:500). Inserts were mounted using ProLong Diamond Antifade Mountant (Invitrogen). Prior to fixation, nuclei were stained with NucBlue Live Cell Stain ReadyProbes according to manufacturer’s instructions (Invitrogen). HepaRG cells were imaged with a Yokogawa CSU-X1 spinning disk confocal system with a Nikon Ti-E inverted microscope using a 60x or 100x Plan Apo objective lens with Zyla cMOS camera using 405, 561 and 488 lasers. NIS elements software was utilized for acquisition parameters, shutters, filter positions and focus control.