Nis element software package

Cryosections (15 μm) on Millennia 1000 slides (StatLab) were fixed for 5 min in 4% PFA in PBS, rinsed three times in PBS, permeabilized in 0.5% Triton-X100/PBS, and incubated with primary antibodies diluted in 10% donkey serum/0.5% Triton X-100 in PBS. Secondary antibodies conjugated to Alexa 594, 488 or 647 (Invitrogen) were incubated in the same conditions. Following DAPI staining, slices were mounted in fluorescent mounting medium (DAKO). Cone arrestin (Millipore) antibody was used in this study. A Nikon Eclipse Ti fluorescence microscope, which was coupled to an LED light source (Lumencor), was used to image the sections with either 20X (n.a. 1.0) or 40X (n.a. 1.4) objectives. The NIS element software package (Nikon) was used for the image acquisition and analysis. The number of cones was counted with the quantification tools from NIS-Elements (Nikon). Cones labelled with specific markers were counted in at least two fields from at least two central retina slices crossing the optic nerve without tracking the orientation of retina. The total number of cones was divided by the length of a reference line drawn on the apical row of the ONL.

Light microscopy images were acquired (single field and z-stacks) with a Nikon Eclipse Ti inverted microscope coupled to an LED light source (Lumencor) and an X-light spinning disk (Crest Optics) with the NIS element software package (Nikon) using 40× (N.A. 1.4) or 100X (N.A. 1.45) objectives. Ependymal cells in Figure 4A were imaged on a Nikon A1Rsi confocal microscope with the 100X (N.A. 1.45) objective.

Image acquisition (single, large scans and Z-stacks) was performed with a Nikon Eclipse Ti coupled to a Coolsnap HQ2 (Photometrics) and an LED light source (Lumencor) with the NIS element software package (Nikon) with either 20× (n.a 1.0) or 40× (n.a. 1.4) objectives. PCs counts were determined with the quantification tools from NIS-Elements (Nikon). PCs labeled with Calbindin were counted across entire cerebellar sections from a minimum of three mice per genotype. The total number of PCs was divided by the length of a line drawn through the PCs layer on each image. The thickness of the GCL was measured at five random locations throughout each image and averaged. A standard t-test was used to compare statistical significance.