Nanozoomer xr slide scanner

Mice were deeply anesthetized and intracardially perfused with PBS (0.1 M) followed by 4% paraformaldehyde (pH=7.4). Thoracic and lumbar spinal cord (SC) sections were removed, post-fixed in 4% paraformaldehyde, and embedded in paraffin. Serial sections with 6 μm thickness were cut, followed by hematoxylin and eosin (H&E) and luxol fast blue (LFB) staining. All reagents were purchased from Sigma (Saint Louis, Missouri, USA). LFB images were captured on an Olympus BX51 multichannel light/epifluorescence microscope (Olympus, Tokyo, Japan). H&E images were captured on a NanoZoomer XR slide scanner (Hamamatsu Photonics, Japan) employing the NanoZoomer Digital Pathology scan software v3.0 (Hamamatsu Photonics, Japan). Density of infiltrating inflammatory cells was determined as the number of cell nuclei per 10.000 µm². The extent of demyelination was evaluated by measuring the percentage of demyelinated area over the total white matter area for each SC section. Quantifications were performed using ImageJ software (NIH, USA).

After immunohistochemistry, the slides were digitalized using the NanoZoomer-XR slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and stored in the J-SHARE archive [30 (link)] in NDPI format. Each whole-slide image was divided into smaller tiles (regions of interest, ROI) that covered the entire image, and single images representing individual cases were captured independently at 40× magnification in JPEG format using NDP.view2 software (Hamamatsu Photonics). Ki-67 protein was analyzed in two steps. First, some captured ROI were evaluated visually by two researchers (T.S. and M.S.). Semiquantitative, 4-grade scoring was applied, ranging from negative (0) to strong (3+) immunoreactivity of the thymocytes. Second, the percentage of Ki-67⁺ thymocytes was determined by automated scoring with Tissue Studio version 3.6.1 software (Definiens, Munich, Germany). Therein, staining thresholds (hematoxylin, 0.07; DAB density, 0.164; medium/low, 0.29; high/medium, 0.45) and morphological filtering (elliptic shape, <0.3; exclusion area, <3; typical nuclear size, 16) were set so that thymocytes within the ROI were recognized in a manner similar to the first analysis. The weight of Ki-67⁺ cells in a thymus was calculated by multiplying the weight of the individual thymus by the percentage of Ki-67⁺ cells in a slide.

The remaining thymus and femur were fixed in 10% neutral buffered formalin. Each femur was decalcified following fixation. These tissues were embedded in paraffin, cut in 1 μm sections, and stained with hematoxylin and eosin for histology. Histology slides were scanned using the NanoZoomer-XR slide scanner (Hamamatsu Photonics, Shizuoka, Japan), and the data were stored in a J-SHARE archive (Japan Storehouse of Animal Radiobiology Experiments), which is an animal-experiment archiving system constructed in our institute [22 (link)]. NanoZoomer data were analyzed using NDP.view2 Plus viewing software (Hamamatsu Photonics, Shizuoka, Japan). The cellularity in the femur was considered from the proportion of total nucleated cells and scored into five categories compared with the cellularity of normal femur as follows: score −4, 0–20% total nucleated cells; score −3, 20–40%; score −2, 40–60%; score −1, 60–80%; score 0, 80–100%. After irradiation, structural changes in the thymic cortex and medulla were also evaluated.