Formalin-fixed specimens of liver and spleen were embedded in paraffin, and sections of 6 µm were cut and mounted on slides (SuperFrost plus, VWR International, Radnor, PA, USA). Tissue slices were stained with Prussian blue (1% potassium ferrocyanide in 1% HCl) and Hematoxylin and Eosin (HE). The histopathological analysis of stained hepatic and splenic tissue sections was carried out on scanned slides (Hamamatsu Nano Zoomer, Hamamatsu Photonics K.K., Hamamatsu, Japan). Grading of steatohepatitis was performed by a veterinary pathologist using a semiquantitative scoring system (scores 1–3) for macrovesicular steatosis, microvesicular steatosis, hypertrophy, and inflammatory foci. For histomorphometric analysis of atherosclerotic plaques, heart and aorta were carefully excised, fixed on a platform, and transferred to formalin. After formalin fixation for 48 h at 4 °C, heart base and aortic root with bifurcations were divided and embedded in paraffin. Sequential sections of 6 µm of the aortic root, aortic arch, and bifurcations were carried out and mounted on slides (see Figure A2 ). Sections were stained with Movat pentachrome staining (Morphisto, Offenbach am Main, Germany), scanned (Hamamatsu Nano Zoomer), and 3 sections of aortic root, aortic arch, and brachiocephalic trunk were analyzed using NDP view software (Version 2.7, Hamamatsu Photonics K.K., Hamamatsu, Japan).
Hamamatsu nanozoomer
Manufactured by Hamamatsu Photonics
Sourced in Germany
The Hamamatsu Nanozoomer is a high-resolution digital slide scanner designed for imaging and analyzing microscopic samples. It captures detailed images of specimens with a high level of clarity and resolution, enabling researchers to conduct thorough examinations and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ndp view software, by Hamamatsu Photonics (1 mentions)
Superfrost plus, by Avantor (1 mentions)
Bond max stainer, by Leica (1 mentions)
Cd30 clone berh2, by Agilent Technologies (1 mentions)
Cd68 clone pg m1, by Agilent Technologies (1 mentions)
Cd20 clone l26, by Agilent Technologies (1 mentions)
Tissuestudio 64, by Definiens (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Stereo investigator software, by MBF Biosciences (1 mentions)
Autostainer xl, by Leica (1 mentions)
Ctm6 coverslipper, by Thermo Fisher Scientific (1 mentions)
Nanozoomer xr, by Hamamatsu Photonics (1 mentions)
Rm2135, by Leica (1 mentions)
6 protocols using hamamatsu nanozoomer
1
Histological Analysis of Liver, Spleen, and Aorta
2
Quantitative Lymph Node Profiling
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For each patient, the whole available tissue specimen/block was cut and subsequent slides were stained in the Department of Pathology at Kiel University for CD3 (clone SP7, Waltham, MA, USA), CD20 (clone L26, Dako, Glostrup, Denmark), CD30 (clone BerH2, maintained at the Department of Pathology, Kiel, Germany) and CD68 (clone PG-M1, Dako, Glostrup, Denmark), using a Leica-Bond-Max stainer (Leica Microsystems, Wetzlar, Germany). The slides were scanned (Hamamatsu Nanozoomer, Hamamatsu Photonics, Ammersee, Germany) and the resulting images were processed by TissueStudio 64, according to the manufacturer’s recommendations (Definiens AG, Munich, Germany). The area ranged between 4-455 mm2 (link) (mean: 133.81 mm2 (link), standard deviation [SD]: 80.84 mm2 (link)) (Online Supplementary Figure S1). Since we included the entire lymph node in the analysis, any heterogeneity of cell distribution did not influence our data. Cutting artifacts, and overstained or unstained areas were manually excluded from the analysis. Adjusting the threshold based on several representative locations on the sample also ensured that the setting for analysis for each sample had been selected to cover the specific staining of the lymph node. See the Online Supplementary Methods for a detailed description and statistics.
3
Quantifying Apoptosis in Newborn and Fetal Mice
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For analysis of cell death in newborn mice, slides were scanned using a Hamamatsu Nanozoomer (Hamamatsu Photonics K.K.) and AC3+ cell quantification was performed using Aperio Image Scope (Leica Biosystems Inc). For the smaller experiments in which cell death was examined in fetuses, AC3 immunostaining was quantified live on a microscope using Stereo Investigator software (MBF Biosciences). In both cases, we drew contours around the areas of interest and AC3+ cells within each contour were quantified as previously described (Ahern et al., 2013 (link); Mosley et al., 2017 (link); Castillo-Ruiz et al., 2018a (link),b (link)). Because AC3 cells are relatively sparse, all positive cells could be counted and random sampling was not required. Cells were counted only when the cell body was clearly visible within the section. The sum of AC3+ cell counts across all sections was divided by total area sampled, and then multiplied by section thickness to obtain the density of AC3+ cells per mm3 for each animal. Investigators blind to experimental conditions performed all analyses.
4
Histological Analysis of Mouse Brain Tissue
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Wild‐type C57BL/6 mice were culled by decapitation and fixed in 4% paraformaldehyde, cryoprotected in a 20% sucrose solution and snap‐frozen in isopentane prior to storage at −80 °C. Sections were cut at 10 μm and stained with H&E using an automatic stainer (Autostainer XL; Leica) and coverslipper (CTM6 Coverslipper; Thermo Scientific, UK). Images were acquired on the LI‐COR Odyssey imager at 700 nm wavelength and by using Hamamatsu Nanozoomer‐XR digital slide scanner. Areas of anatomical interest were identified and measured by drawing around each region using either imagej software on the sections scanned with the Hamamatsu Nanozoomer or image studio lite (version 5) software with sections scanned using the LI‐COR Odyssey IR imager.
5
Quantifying Corneal Inflammatory Cell Infiltration
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Eyes were fixed in 2% paraformaldehyde overnight before being processed and embedded in paraffin wax. Thereafter, tissue was cut into 5-μm thick sections using a microtome (Leica RM 2135) and left overnight at 37 °C. The sections then were deparaffinized in xylene (twice for 5 minutes) and rehydrated in a gradient of ethanol (100%, 95%, and 75%, all twice for 5 minutes) and stained with hematoxylin–eosin to identify infiltrating cells. Slides were scanned digitally at ×20 magnification using a Hamamatsu Nanozoomer (Hamamatsu Photonics) in brightfield mode (OracleBio; BioCity). Infiltrating cells were counted in the corneal stroma region of interest with NanoZoomer Digital Pathology. Version 2.0, viewer software (Hamamatsu Photonics) by a masked investigator to avoid bias. Counted cells were normalized to area (square millimeters).
6
Multimodal Mapping of Subthalamic Area
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Adjacent series of regularly spaced sections (1/10, 500 µm) of human and monkey brains were immunohistochemically processed for tyrosine hydroxylase (TH), orexin A (OX), anti-serotonin transporter (SERT), vesicular glutamate transporter 1 (VGluT1), choline acetyl transferase (ChAT), and oestrogen receptors of α type (ERα) and were incubated with secondary antibodies from the appropriate species (see Supplementary method 1 for details). Some sections were counterstained with cresyl violet. All sections were digitised using a Hamamatsu Nano Zoomer (Hamamatsu Photonics, France).
The subthalamic area was delineated by the subthalamic nucleus laterally, the zona incerta dorsally, the ventral tegmental area ventrally, the posterior hypothalamus anteriorly, and the wall of the third ventricle medially. Within the subthalamic area, the MFB was delineated using TH and SERT immunostained bres and the Sano triangle using adjacent structures such as the mammillothalamic tract laterally and the third ventricle medially, the zona incerta dorsally and the ventral tegmental area ventrally, the mammillary body anteriorly and the red nucleus posteriorly (Supplementary Fig. 1).
The subthalamic area was delineated by the subthalamic nucleus laterally, the zona incerta dorsally, the ventral tegmental area ventrally, the posterior hypothalamus anteriorly, and the wall of the third ventricle medially. Within the subthalamic area, the MFB was delineated using TH and SERT immunostained bres and the Sano triangle using adjacent structures such as the mammillothalamic tract laterally and the third ventricle medially, the zona incerta dorsally and the ventral tegmental area ventrally, the mammillary body anteriorly and the red nucleus posteriorly (Supplementary Fig. 1).
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