Tcs scp8

The paraformaldehyde-fixed and sucrose-cryoprotected frozen right hemispheres from wt and NPC1 mice were used to prepare 10 μm thick sagittal cryosections (Leica CM 3050S cryotome, Wetzlar, Germany). For immunohistochemistry, cryosections were dried in the hood for 1 h, permeabilized in TBS-T (50 mM TBS, pH 8.0, 0.5% Triton X-100, Sigma-Aldrich, St. Louis, MO, USA) for 30 min and blocked in 4% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) in TBS-T for 1 h. The sections were subsequently incubated with primary antibodies diluted in 4% donkey serum in TBS-T overnight. After a 3 h incubation with secondary antibody anti-rabbit-Alexa488, anti-mouse-Alexa594 or anti-goat-Alexa647 (from donkey, Molecular Probes, Invitrogen, Waltham, MA, USA) and Hoechst to counterstain nuclei (Sigma-Aldrich, St. Louis, MO, USA), sections were mounted (Fluoromount, Sigma-Aldrich, St. Louis, MO, USA). Confocal images were acquired on an inverted laser scanning confocal microscope Leica TCS SCP8 with the software LAS X (Leica, Wetzlar, Germany), and additional image processing and quantification were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cortical neurons were isolated from postnatal day 0 (P0) mouse pups and grown as described previously [24 (link)]. Neurons were plated on poly-L-lysine (0.5 mg/mL, Sigma Aldrich) coated glass coverslips (Marienfeld) and grown in Neurobasal medium complemented with 2% B27 and 2mM L-glutamine (all from Gibco, Thermo Fisher Scientific). Immunocytochemistry on cortical mouse neurons was performed as previously described [14 (link)]. In short, cortical neurons grown on coverslips were fixed with 4% sucrose/paraformaldehyde for 15 minutes, quenched in 50mM ammonium chloride and permeabilized with 0.1% Triton X-100 for 3 minutes. The neurons were blocked at room temperature for 1 hour in 5% goat serum (Sigma Aldrich). Neurons were subsequently incubated with primary antibodies diluted in blocking solution overnight, followed by secondary antibody incubation (Alexa Fluor 488, 594 and 647, Thermo Fisher Scientific) together with filipin (Sigma Aldrich, as described by Neufeld et al. 1999). Confocal images were acquired on an inverted laser scanning confocal microscope Leica TCS SCP8. Images were acquired with the software LAS X (Leica) and additional image processing and were performed using ImageJ software (National Institutes of Health, USA).