Nanozoomer microscope

MS grey matter brain lesions were staged on the basis of demyelination criteria, as described earlier [14 (link)]. Grey matter tissue was stained with monoclonal antibodies against PLP, NeuN and MAP2 using immunohistochemical detection. The slices were scanned using a Nanozoomer microscope (Hamamatsu). Lesions were digitally staged according to the classification scheme for grey matter demyelinating lesions, using the Nanozoomer Digital Pathology software version 1.2. Shortly, lesions were scored as mixed white-grey matter (Type I), intracortical (Type II), subpial (Type III) and lesions stretching from the pia mater until the border of white matter (Type IV). Two independent observers (KLK, MvM), blinded to clinical information and kif21b expression, scored grey matter lesions based on PLP staining, on a standardized scoring form. Morphometric information regarding total area of the slide, area containing white matter in the tissue (based on absence of MAP2 and NeuN expression), total number, types and area of grey matter lesions was assessed. Moreover, expression of HLA-II was scored as well as meningeal infiltration. In addition, when the slide contained white matter lesions, the area and HLA-II expression of these lesions was scored.

Tranplanted host eyes (N = 6) were processed and cryosectioned as described for the Y chromosome FISH experiment, and subsequently stained for GFP (1:500; Abcam, ab13970) and photoreceptor specific marker RCVRN (1:5000; Millipore, AB5585), followed by secondary antibody staining (1:1000; Jackson Immunoresearch) Every fourth serial section from whole experimental retinas was used to quantify the total amount of YFP⁺ photoreceptors. Cells were counted from images obtained with the NanoZoomer microscope (Hamamatsu Photonics). Following these cell counts, the resulting value was multiplied by four to estimate the total amount of labelled cells per retina.
For information on antibodies used, see Supplementary Table 4.

Brain sections were mounted and allowed to dry overnight. The following day, sections were stained in cresyl violet for 5 min at room temperature. The slices were then sequentially dehydrated in increasing ethanol concentrations followed by xylene and coverslipped with Cytoseal 60 (8310-16; Thermo Fisher Scientific). Slides were scanned using Hamamatsu’s Nanozoomer microscope at 20X magnification. Hippocampus and entorhinal/piriform cortex were traced using the NDP view 2. Volumetric analysis of the hippocampus and the entorhinal/piriform cortex was performed via stereological methods by assessing sections spaced 300 μm starting from bregma −1.2 mm to bregma −3.3 mm (6–8 sections per mouse depending on the severity of brain atrophy). The formula for the volumetric calculation was volume = (sum of area) ∗ 0.3 mm. Three sections, corresponding approximately to bregma coordinates −2.1, −2.4, and −2.7 mm were selected to measure the thickness of the dentate gyrus granular cell layer by drawing a scale perpendicular to the cell layer in all three slices and taking the average value for each mouse.

Immunofluorescence was observed using a Leica DM6000 microscope (Leica microsystems) equipped with a CCD CoolSNAP-HQ camera (Roper Scientific) or using an inverted or upright laser scanning confocal microscope (FV1000, Olympus) with 405, 488, 515 and 635 nm pulsing lasers. The images were acquired sequentially with the step size optimized based on the Nyquist–Shannon theorem. The analysis was conducted in FIJI (NIH). Images were put into a stack, Z-sections were projected on a 2D plane using the MAX intensity setting in the software’s Z-project feature, and the individual channels were merged.
Images of Y chromosome labelled retinas were acquired using SIM (ApoTome, Zeiss). Samples stained to perform quantification of surviving YFP⁺ photoreceptors were imaged with the NanoZoomer microscope (Hamamatsu Photonics).