Hcx pl apo 10 0.40 cs

Images of tissue sections were acquired with a confocal laser scanning microscope (TCS SP8; Leica Microsystem) using a 5× air (HCX PL Fluotar 5×/0.15, numerical aperture [NA] = 0.15, Leica Microsystems), 10× air (HCX PL APO 10×/0.40 CS, NA = 0.40, Leica Microsystems), 16× multi-immersion (HC FLUOTAR 16×/0.60 IMM CORR VISIR, NA = 0.60, Leica Microsystems) and 25× water-immersion (HC FLUOTAR L 25x/0.95 W VISIR, NA = 0.95, Leica Microsystems) objective lens. The confocal pinhole was set to 1.0 to 5.0 Airy unit. Confocal z-stack images were collected at 1.0 to 30 μm intervals at 512 × 512 or 1024 × 1024 pixel resolution. Acquired images were tiled, stitched and processed to create maximum intensity projection using Leica Application Suite X software (LAS X, ver. 3.5.5.19976, Leica Microsystems) and ImageJ software^{60 (link)} (ver. 1.53, National Institutes of Health). The global brightness and contrast of the images were adjusted with ImageJ software.

3D image stacks were acquired with a TCS SP8 CLSM (Leica Microsystems). A 16× multi-immersion objective lens (HC FLUOTAR 16×/0.60 IMM CORR VISIR, numerical aperture [NA] = 0.60, WD = 2.5 mm, Leica Microsystems) was used for imaging the optically cleared brain slices (1-mm thick). 10× air (HCX PL APO 10×/0.40 CS, NA = 0.40, WD = 2.20 mm, Leica Microsystems), 20× multi-immersion (HC PL APO 20×/0.75 IMM CORR CS2, NA = 0.75, WD = 0.66 mm, Leica Microsystems), 25× water-immersion (HC FLUOTAR L 25×/0.95 W VISIR, NA = 0.95, WD = 2.40 mm, Leica Microsystems), and 63× oil-immersion (HC PL APO 63×/1.40 Oil CS2, NA = 1.40, WD = 0.14 mm, Leica Microsystems) objective lenses were used for imaging the re-sections (40- or 50-μm thick). Sections were mounted with PBS or 75% glycerol in PBS (Hirabayashi et al., 2018 (link)). DAPI, EGFP, and mScarlet were excited by 405-, 488-, and 552-nm lasers, and their emissions were collected through 410–480, 495–525, and 560–700 nm emission prism windows, respectively.