Hc pl apo 63x 1.40 oil cs2

For confocal microscopic co-localization studies, the cells were seeded onto BD Falcon Cultures Slides (Becton Dickinson). Next day the fibroblasts were washed, fixed with ice-cold methanol, blocked with 5% FBS in PBS and co-labelled rabbit monoclonal anti-NPC1 (1:100, ab134113, Abcam) and mouse monoclonal anti-LAMP2 (1:500, H4B4, Iowa Hybridoma Bank) antibodies at 4 °C overnight. Secondary antibodies were donkey anti-IgG anti-mouse alexafluor555 or anti-rabbit alexafluor488 conjugates (Pierce) diluted 1:1000. Leica SP8X laser scanning confocal system equipped with 470 nm–670 nm 80 MHz pulse continuum White Light Laser 2 and HC PL APO 63x/1.40 OIL CS2 (W.D. 0.14 mm) objective was used to image the samples. Image acquisition conditions were: excitation at 496 nm or 553 nm, one voxel 42.2 × 42.2 × 130 nm, 7 Z-steps (fulfilling Nyquist sampling theorem), Hybrid detectors at 503–553 nm or 566–650 nm. Acquired confocal images were deconvolved using theoretical point spread function in Huygens Professional software (Scientific Volume Imaging - SVI, Hilversum, The Netherlands). Overlay colocalization maps and Object Pearson’s coefficients were computed using Huscript (SVI), the grayscale maps were converted to colour coding look-up table (LUT) in Fiji/ImageJ software (NIH, Bethesda).