Zen 2010

Images were acquired using a Zeiss LSM 700 inverted confocal microscopy (Zeiss) using a Plan-Apochromat 60x/1.4 NA oil objective and acquired using Zen 2010 software (Zeiss). For any given experiment, the same scan speed and laser power were used to ensure an accurate comparison between cells and conditions. Quantisation of fluorescence intensity and correcting the brightness and contrast of the images was performed with ImageJ software (NIH) or Zen 2010 software (Zeiss). Quantification of the plasmalemmal: cytoplasm fluorescence for the Lact-C2 and D4H probes was performed using the region of interest tool in ImageJ. First, mean pixel intensities for the plasma membrane and cytoplasm were calculated for each cell. Next, the quotient of these values was obtained to determine an enrichment of probe in the PM. For display panels, post-acquisition adjustments were made homogenously across the entire image, and the linearity of mapped pixel values was not altered.

For fluorescence microscopy, cells were grown on a coverslip to 50% confluency. For lysosomal staining, cells were incubated with 0.5 μmol/L Lysotracker Red DND-99 (Life Technologies; L7528) in full medium for 45 minutes before fixation. Cells were fixed with 4% PFA in phosphate-buffered saline for 10 minutes at room temperature. For determination of neutral lipids enclosed in cytosolic lipid droplets, cells were incubated with HCS LipidTOX Green neutral lipid stain (Life Technologies; H34475) according to manufacturer’s protocol. For imaging, cells were mounted with Vectaschield DAPI stain (Vector Laboratories, Burlingame, CA; H-1000-10) according to manufacturer’s protocol for nuclear staining. Cells were imaged at room temperature on a Zeiss (Jena, Germany) Observer Z1 brightfield microscope equipped with ApoTome 2 and a 63× (W) objective lens (APO DIC III numerical aperture 1.2) and acquired using Zeiss software Zen 2010. Laser lines used in this study were 405, 488, and 568 nm. Red/green/blue and greyscale images were further processed with Zeiss software Zen 2010. In addition, HepG2 cells and HLCs were used to assess cellular and surface LDL receptor protein expression levels with Western blot and flow cytometry and LDL uptake with Dylight labeled LDL particles in combination with flow cytometry.²⁴

We imaged the dorsal air sac primordium (ASP) from third instar larvae (Sato and Kornberg, 2002 (link)). Slides were imaged using a Zeiss LSM 710 (Carl Zeiss Ltd) laser scanning confocal system with an inverted Axio Observer.Z1 microscope. Zeiss Fluor 20×/0.75 air and 40×/1.3 oil immersion objectives were used. GFP fusion proteins were excited at 488 nm, using an Argon laser and detected maximally at 509 nm. mRFP fusion proteins and Alexa Fluor 555 were excited at 543 nm using a He/Ne laser and detected maximally at 607 and 565 nm, respectively. The pinhole was set at ∼1 AU. When z-stacks were taken, we used the slice thickness specified by the software for 1 AU (usually 0.5–2 µm). Images were captured using Zen 2010 (Zeiss) software, exported in tagged image file format (TIFF) and edited in Adobe Photoshop CS5 (Adobe Systems Europe Ltd, Maidenhead, UK). When z-stacks were produced, images are presented as single slices, unless a projection is specified. When a 3-dimensional (3D) projection was required, stacks were rendered using Zen 2010 software (Zeiss). If image brightness was altered for publication, this change was standardized across groups to retain comparability.