Liver tissue samples collected from F0/F1, F3, and F7 dams were sent to the Institute of Veterinary Pathology at the University of Zurich for further evaluation. Liver tissue was embedded into a paraffin block, sliced in 3–5 μm thick sections and mounted onto glass slides. Following hematoxylin and eosin-staining (H&E), slides were scanned using a slide scanner (NanoZoomer-XR C12000; Hamamatsu, Hamamatsu City, Japan). Liver sections were semi-quantitatively scored using the non-alcoholic fatty liver disease (NAFLD) Clinical Research Network Scoring System (Kleiner et al., 2005 (link)), taking into account that NAFLD in rodents is not associated with the development of megamitochondria and a few other features described in human NAFLD (Liang et al., 2014 (link)). Under this steatosis scoring system, a score of 0 represents not present, 1 represents very mild, 2 represents mild, 3 represents moderate, and 4 represents severe steatosis.
Nanozoomer xr c12000
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer-XR C12000 is a high-resolution digital slide scanner designed for digitizing glass slides. It captures images at a maximum resolution of 0.23 μm/pixel, enabling detailed visualization of microscopic samples. The system utilizes a line-scanning approach to rapidly digitize entire slides. Key features include automated slide loading, high-speed image capturing, and compatibility with a variety of slide sizes.
Automatically generated - may contain errors
Lab products found in correlation
Ndp view2, by Hamamatsu Photonics (3 mentions)
Integrator system, by Visiopharm (3 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Anti pd l1 antibody, by Cell Signaling Technology (1 mentions)
Anti cd8 antibody, by Nichirei Biosciences (1 mentions)
Ab20034, by Abcam (1 mentions)
Cryostat, by Leica (1 mentions)
Storm phosphorimager, by GE Healthcare (1 mentions)
Phosphate buffered saline (pbs), by Wisent (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
View2, by Hamamatsu Photonics (1 mentions)
Matrigel, by Corning (1 mentions)
Formaldehyde neutral buffer solution, by Fujifilm (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Formalin, by Nacalai Tesque (1 mentions)
Rem 710, by Yamato Scientific (1 mentions)
20 protocols using nanozoomer xr c12000
1
Histological Evaluation of Rodent Fatty Liver
2
Immunohistochemical Profiling of Tumor Microenvironment
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All TMA sections were stained as follows. FFPE samples were cut into 4–5 μm thick sections and placed onto silane-coated glass slides. After deparaffinization, the sections were processed for antigen retrieval. After blocking, the sections were incubated overnight with primary antibodies, followed by incubation with secondary antibodies conjugated to a peroxidase-labeled dextran polymer. The primary antibodies used were as follows: anti-Bcl6 antibody (#418181, Nichirei., Tokyo, Japan), anti-CD3 antibody (#413591, Nichirei., Tokyo, Japan), anti-CD8 antibody (#413211, Nichirei., Tokyo, Japan), anti-CD10 antibody (#413261, Nichirei., Tokyo, Japan), anti-CD20 antibody (#422441, Nichirei., Tokyo, Japan), anti-CD21 antibody (#M0784, Dako), anti-FOXP3 antibody (to label Treg cells, #ab20034, Abcam), anti-PD-1 antibody (#43248, Cell Signaling Technology), and anti-PD-L1 antibody (#13684, Cell Signaling Technology). Immunohistochemical staining was visualized using 3,3’-diaminobenzidine in 50 mM Tris-HCl (pH 5.5) containing 0.005% hydrogen peroxidase. Finally, the sections treated with 3,3’-diaminobenzidine were counterstained with hematoxylin. All stained TMA sections were scanned and visualized using a high-resolution digital slide scanner (NanoZoomer-XR C12000; Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan).
3
Autoradiography for Tumor Quantification
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For autoradiography, tumors were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA) and frozen at −20 °C. Every 200 μm, a 20 μm section was cut with a cryostat (Leica Microsystems, Wetzlar, Germany) at −19 °C. A storage phosphor screen was placed on the slices and read out after an exposure time of 10 half-lives of the respective tracer with a pixel size of 50 μm using a STORM Phosphor-Imager (Molecular Dynamics, Sunnyvale, CA, USA). Tissue slices were then stained with hematoxylin and eosin (H&E), and whole-slide images were digitized using a digital slide scanner (NanoZoomer-XR C12000, Hamamatsu Photonics K.K., Hamamatsu-City, Japan). For normalization, autoradiography was analyzed as tumor-to-muscle-ratios (TMR), dividing the whole autoradiography plate of each mouse by the mean value of the muscle samples on the plate (ImageJ; National Institute of Health, Bethesda, USA45 (link)).
4
Stereological Analysis of Mouse Lung
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Lung structure was assessed by design‐based stereology with systemic uniform random sampling, on mouse lungs that were pressure fixed at 20 cm H2O, and treated with arsenic, osmium and uranium, and embedded in plastic (Technovit 7100) resin, sectioned at 2 μm, stained with Richardson's stain, and image captured in a Nanozoomer‐XR C12000 (Hamamatsu), exactly as described previously (Madurga et al, 2014 ; Mižíková et al, 2015 ; Nardiello et al, 2017b ). Lung volume was determined by the Cavalieri principle (Madurga et al, 2014 ). Stereological analyses were undertaken using the NewCast PLUS version VIS4.5.3. computer‐assisted stereology system (Visiopharm) and facilitated the determination of inter alia total number of alveoli in the lung, the mean septal thickness, and total gas‐exchange surface area.
5
Digitizing Research Slides with Precision
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Slides were scanned with a NanoZoomer-XR C12000 (Hamamatsu) and scans were processed in NPDI.Viewer2 (Hamamatsu).
6
High-Resolution Digital Slide Scanning
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We acquired color images using a Hamamatsu NanoZoomer-XRC12000 high-resolution digital slide scanner (Hamamatsu City, Japan).
7
Histopathological Analysis of Larval Tissues
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The larvae at each time point were subjected to histopathological examination (Supplementary File S13 ) to study the tissue level changes (Perdoni et al., 2014 (link)). The microscopic visualization was performed (Leica Microscope DMLB) and the image acquisition was carried out (NanoZoomer-XR C12000, Hamamatsu Photonics, Japan).
8
Histological Analysis of Spinal Cord
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Mice were euthanized, perfused with ice-cold PBS (Wisent, St. Bruno, QC, Canada), and the spinal cords were removed and fixed in 4% formaldehyde for 24 hours. T5-μm sections of lumbar spinal cord were used for hematoxylin–eosin (HE) or immunofluorescence staining of the following markers: CD3 (Abcam, Cambridge, MA, ab5690), GFAP (Cell signaling Technology, Danvers, MA,12389), Iba1 (Wako, Osaka, Japan, 019–19741), MBP (Abcam, Cambridge, MA, ab40390), or IgG (Cell signaling Technology, Danvers, MA, 4408). All slides were scanned using a digital slide scanner NanoZoomer-XR C12000 (Hamamatsu, Hamamatsu City, Japan) and viewed using NDPview2 software (Hamamatsu). The percentage of positive area of the stained markers was quantified after thresholding of images using Fiji (ImageJ) software (NIH). Antibody dilutions were prepared based on the manufacturer’s instruction.
9
Quantifying DNA Damage in Tumor Cells
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The number of DNA DSBs induced in AR42J tumor cells after exposure to the radiopeptides was assessed by immunostaining of γH2AX. The cells were treated in Petri dishes for 2 h using 2.5 MBq/mL or 10 MBq/mL of each radiopeptide followed by incubation with fresh culture medium for additional 24 h. Cell pellets obtained after centrifugation were fixed and embedded in paraffin and cut into sections. The immunostaining was performed using a phospho-histone H2A.X (Ser139) rabbit monoclonal antibody (Cell Signaling Techonology, Danvers, Massachusetts, USA) and an anti-rabbit, horseradish peroxidase-derivatized secondary antibody with DAB substrate buffer (Agilent Technologies, Santa Clara, California, USA) (Supplementary Material ). The sections were scanned using a digital slide scanner (NanoZoomer-XR C12000; Hamamatsu, Japan) and the positively and negatively stained AR42J tumor cells were quantified with the pathology image analysis software VIS (Visiopharm Integrator System, Version 208 2019.02.2.6239, Visiopharm, Hoersholm, Denmark) (Supplementary Material ). The percentage of γH2AX-positive cells in sham-treated samples was in average 1%. Data were analyzed with a one-way ANOVA with Dunnet’s multiple comparisons post-test.
10
Quantifying Pulmonary Inflammation via Immunofluorescence
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Lungs from treated mice were harvested and fixed in 4% formaldehyde for 24 hours and kept in ethanol for analysis. Lungs were then embedded in paraffin and 5 µm sections were used for immunofluorescence staining using CD3, IFNγ and granzyme B. All antibodies are listed in online supplementary table S1 . Slides were scanned using a digital slide scanner (Nanozoomer-XR C12000; Hamamatsu) provided by the Histology Platform (Université de Sherbrooke). Percentage staining of marker-positive areas were quantified using ImageJ software (NIH).
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