Ix83 inverted imaging system

For immunhistochemical staining of HAP1 cells, culture media was aspirated and cells were washed once with 1× PBS. Cells were fixed with ice-cold 4% paraformaldehyde in 1× PBS containing 15% sucrose for 20 min at room temperature (RT) and subsequently permeabilized with 0.1% Triton X-100 in 1× PBS for 3 min at 4 °C. For F-actin staining, Phalloidin-iFluor 594 (Abcam, Cambridge, United Kingdom) was diluted 1 : 1000 in 1% bovine serum albumin (Carl Roth, Karlsruhe, Germany) in 1× PBS and for staining of cell nuclei 0.5 μg mL⁻¹ DAPI (Carl Roth) was added. Cells were incubated for 1 h at RT, washed with 1× PBS and coverslips were mounted on microscope slides with Shandon Immu-Mount mounting media (Thermo Fisher Scientific).
Bright-field images of printed HAP1 and HEK293H cells were captured with a CKX53 inverted microscope with integrated Phase Contrast (iPC) and a XM10 monochrome camera (Olympus, Shinjuku, Japan). Fluorescence images were acquired with an IX83 inverted imaging system with a DP80 camera (Olympus) and a 4-channel high-specification LED System (Judges Scientific, London, United Kingdom). Olympus cellSense software was used with both microscopes, and adjustments of brightness and contrast were carried out with ImageJ (NIH, Bethesda, MD, USA).

Brightfield images of ISH and fluorescence images of immunohistochemical stainings of brain slices were captured with an IX83 inverted imaging system with a DP80 camera (Olympus, Shinjuku, Japan). Images were taken with either 20× UPlanSApo (0.75 NA) or 60× UPlanSApo (oil‐immersion, 1.35 NA) objectives. Confocal images of primary neurons and brain slices were acquired with upright laser microscopes (Leica DM 2500 and Leica SP8, Leica Microsystems, Wetzlar, Germany) equipped with a 63× objective (oil‐immersion, 1.2 NA) using sequential scanning with the 488‐nm line of an argon‐ion laser and the 543‐nm line from helium‐neon lasers (for Alexa 488 and Alexa 568, respectively). Background correction and adjustment of brightness and contrast were performed using either Leica confocal software (Leica Microsystems, Wetzlar, Germany), cellSens software (Olympus, Shinjuku, Japan), or ImageJ (NIH, Bethesda, MD, USA).

NPC1 patient-specific fibroblasts (passage 5 of primary fibroblasts from sibling 1 and sibling 2) and fibroblasts of an apparently healthy individual (control: GM08398) were seeded at a density of about 25,000 cells/cm² on PLL-coated coverslips and cultivated for 24 h at 37 °C and 5% CO₂. Afterwards, cells were fixed with 4% PFA (Merck KgaA) dissolved in 1x PBS for 15 min at RT, washed 2 × 5 min in 1x PBS and stored in 0.02% sodium azide (NaN3) at 4 °C. Cells were incubated in 0.1-mg/mL Filipin (Polysciences, Inc., Warrington, PA, USA) for 45 min, mounted with Mowiol-DABCO Mounting medium (Carl Roth GmbH) and used for microscopy (Figure S5). Differential interference contrast (DIC) images and fluorescence images were captured using the IX83 inverted imaging system with a DP80 camera (Olympus, Shinjuku, Japan). Images were taken with a 60x UplanSApo (oil-immersion, 1.35 NA) objective using the Olympus cellSens software (Olympus). Background correction and adjustment of brightness and contrast were performed using ImageJ (NIH, Bethesda, MD, USA).