We used NanoZoomer RS slide scanner with constant light intensity and exposure time and NanoZoomer Digital Pathology System view software (Hamamatsu, Hamamatsu, Japan). To quantify SCI and GW2580 treatment-induced changes in IBA1 expression, the mean optical density (OD) was measured along the spinal cord, as previously described (ImageJ, National Institutes of Health, USA) 23 (link), 33 (link), 34 (link), 37 . To minimize bias in staining intensity, all immunostainings for a given antibody and a given time-point were done in parallel. For all antibodies used, expression levels were analyzed in at least 40 and 16 axial sections throughout the lesion segment of the spinal cord at 210 µm and 630 µm intervals for Microcebus Murinus and mice respectively. OD quantifications included grey and white matters (excluding the dorsal funiculus) and dorsal funiculus. Background was subtracted from OD values of each section. All quantifications were done blindly.
Nanozoomer rs slide scanner
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer RS is a high-resolution slide scanner designed for digital pathology applications. It captures high-quality images of microscope slides with a maximum resolution of 0.23 microns per pixel. The scanner uses a line-scanning technology to digitize the entire slide efficiently.
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Lab products found in correlation
Nanozoomer digital pathology system view software, by Hamamatsu Photonics (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Dm1000 led, by Leica (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Rnase a, by Qiagen (1 mentions)
Ndp view2, by Hamamatsu Photonics (1 mentions)
4 protocols using nanozoomer rs slide scanner
1
Quantifying IBA1 Expression in SCI
2
Standardized Imaging Protocols for Reproducible Analysis
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All images were obtained using NanoZoomer RS slide scanner that uses constant light intensity and exposure time (NanoZoomer Digital Pathology System and NDP view software, Hamamatsu City, Japan). NDP view software was used to export images at a constant magnification. All images were exported with the same parameters for all sections in each immunohistochemistry and time points. All quantifications were done blindly. High magnifications were obtained using a bright-field up-right microscope (Leica DM1000 LED, Mannheim, Germany). For immunofluorescence images, we used laser scanning confocal microscopy (Leica SPE, Mannheim, Germany). Laser intensity and detector sensitivity settings were kept constant for all image acquisitions within a given experiment.
3
Optimizing DNA Extraction from FFPE Tissues
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DNA extraction was performed using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) for FFPE tissues, the QIAamp DNA Mini Kit (Qiagen) for frozen tissues, and the DNeasy Blood & Tissue Kit (Qiagen) for cultured cells, according to the manufacturer's protocols. Each FFPE tissue block was cut into 5 μm thick scroll sections, and 2 to 10 sections were put into centrifuge tubes, to obtain a tissue volume between 2 and 5 mm 3 . Xylene was used for deparaffinization following the manufacturer's protocol. RNase A (Qiagen) was used for excluding copurified RNA. For the comparative study on the influences of tissue section thickness and prolonged proteinase K digestion on DNA quality, FFPE blocks were sliced to thicknesses of 1 μm (20 sections) and 10 μm (2 sections), and proteinase K digestion was prolonged from 1 h (manufacturer's recommendation) to 24 h and 48 h. DNA concentration and purity were measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, USA). DNA yields were calculated using DNA amounts and tissue volumes of FFPE blocks. Tissue volumes were calculated using section thickness and surface area measured using whole slide images of hematoxylin and eosin stained sections, obtained using the NanoZoomer-RS slide scanner with NDP.view2 software (Hamamatsu Photonics, Japan).
4
Spinal Cord Injury Microscopy Analysis
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Brightfield microscopy: We used NanoZoomer RS slide scanner with constant light intensity and exposure time and NanoZoomer Digital Pathology System view software (Hamamatsu, Hamamatsu, Japan). To quantify SCI and GW2580 treatment-induced changes in IBA1 expression, the mean optical density (OD) was measured along the spinal cord, as previously described (ImageJ, National Institutes of Health, USA) [22, 57, 58, 61] . To minimize bias in staining intensity, all immunostainings for a given antibody and a given time-point were done in parallel. For all antibodies used, expression levels were analyzed in at least 40 and 16 axial sections throughout the lesion segment of the spinal cord at 210µm and 630µm intervals for Microcebus Murinus and mice respectively. OD quantifications included grey and white matters (excluding the dorsal funiculus) and dorsal funiculus. Background OD was subtracted from OD values of each section. All quantifications were done blindly. μs/scan, respectively) and either a zoom x 1.2 or x 3. CARS excites the CH2 vibrational mode at 2845cm -1 and CH2 bonds are found in lipids [64] . Excitation wavelengths are 836 and 1097nm (synchronized Ti-saphire and OPO, respectively) and the signal is detected at 675nm (filter from 660-685nm). Pictures are a stack of 3µm (3 slices). Myelin degradation and myelin density were scored and manually quantified, respectively.
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