For counts from units expressing neck, chief, and proliferative markers all time points were quantified with at least 3 mice. Stomach corpus slides were stained for 5-bromo-2′-deoxyuridine (BrdU), the neck cell marker GSII lectin, and zymogenic cell marker GIF. Images were captured as TIFF files from a Zeiss Axiovert 200 microscope with an Axiocam MRM camera with an Apotome optical sectioning filter (Carl Zeiss, Jena, Germany). Each stomach had at least five randomly distrubuted 20× images taken, which contained 10 or more well-oriented gastric units. Units were counted using the neck staining, and total quantifications of proliferating cells (5-bromo-2′-deoxyuridine) were averaged over the total unit numbers per mouse. For total proliferation counts from immunohistochemistry at least 3 mice per treatment group were quantified. Stomach corpus slides were stained with an antibody for 5-bromo-2′-deoxyuridine (BrdU). Whole slides were scanned using a NanoZoomer 2.0 HT microscope and analyzed with NanoZoomer Digital Pathology software (Hamamatsu; Hamamatsu City, Japan). At least 40 corpus units were analyzed and counted per stomach. Proliferation rate for each mouse was an average of all units counted.
Nanozoomer digital pathology software
Manufactured by Hamamatsu Photonics
Sourced in Japan
The NanoZoomer Digital Pathology software is a digital slide scanning and management system developed by Hamamatsu Photonics. It is designed to capture high-quality digital images of microscope slides and manage the resulting digital files. The software provides tools for scanning, viewing, and analyzing digital slides.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (6 mentions)
Anti perilipin, by Abcam (2 mentions)
Nanozoomer slide scanner system, by Hamamatsu Photonics (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Nanozoomer 2.0 rs scanner, by Hamamatsu Photonics (2 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Fluorescently conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Zen digital imaging for lsm 700, by Zeiss (1 mentions)
Zen 2010b sp1 imaging software, by Zeiss (1 mentions)
Anti β tubulin, by Novus Biologicals (1 mentions)
Nod scid gamma male mice, by Jackson ImmunoResearch (1 mentions)
Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)
Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions)
20 protocols using nanozoomer digital pathology software
1
Gastric Corpus Proliferation Quantification
2
Xenograft Tumor Formation from Human iPSCs
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Human iPSCs cultured in 6-well plates were dissociated with DPBS containing 0.5 mM EDTA and approximately 1 × 107 dissociated cells were collected and resuspended in 400 μl culture medium supplied with 25 mM HEPES (pH 7.4) and stored on ice. Then, 50% volume (200 μl) of cold Matrigel (354277, Corning) was added and mixed with the cells. The mixture was injected subcutaneously into NSG mice (JAX No. 005557) at 150 μl per injection site. Visible tumors were removed 6–8 weeks post injection, and were immediately fixed in 10% Neutral Buffered Formalin. The fixed tumors were embedded in paraffin and stained with hematoxylin and eosin. Images were collected using the NanoZoomer Digital Pathology software (Hamamatsu).
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.scr.2019.101461 .
Supplementary data to this article can be found online at
3
Oxidative Stress Assay on HUVECs
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10,000 cells/well (400 µL of cell suspension in complete DMEM) were seeded in a Lab-Tek and incubated for 48 h. HUVECs were then washed with 100 µL PBS, added by 175 µL of fresh complete DMEM and treated with 13 µL of PBS (healthy HUVECs) or 13 µL of 150 µM AM (oxidatively stressed HUVECs) for 1 h at 37 °C in the dark. Cells were washed with 100 µL PBS, added by 175 µL of fresh complete DMEM and incubated with 25 μL of samples (AX/NHs or NHs in PBS) at the final concentration of 250 μg mL−1 and incubated for 24 h. For an appropriate comparison, cells received 25 μL of PBS. HUVECs were washed with 400 µL PBS, fixed with 200 µL 4% PFA for 30 min at 4 °C, washed twice with 400 µL PBS and added by 200 µL of 0.1% Triton for 5 min at 25 °C. Cells were washed again twice with 400 µL PBS and then treated with 200 µL DAPI (dilution 1:1000) and phalloidin (dilution 1:200) solution for 30 min at 25 °C. Cells were finally washed twice with PBS (400 µL), slides were fixed and then digital images were obtained and analysed using Nanozoomer digital pathology software (Hamamatsu, Japan).
4
Quantitative Immunohistochemistry for β-cell Mass
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Immunohistochemistry analyses and β cell mass were performed as described previously (60 (link)). Images were captured using a NanoZoomer slide scanner system with NanoZoomer Digital Pathology software (Hamamatsu) or confocal fluorescence microscopy. Microscopic images were analyzed using ImageJ software.
5
Teratoma Formation from Patient iPSCs
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Patient iPSCs cultured in 6- well plates were dissociated with DPBS containing 0.5 mM EDTA and approximately 1 × 107 dissociated cells were collected and re-suspended in 400 μl culture medium supplied with 25 mM HEPES (pH 7.4) and stored on ice. Then, 50% volume (200 μl) of cold Matrigel (354277, Corning) was added and mixed with the cells. The mixture was injected subcutaneously into NSG mice (JAX No. 005557) at 150 μl per injection site. Visible tumors were removed 6–8 weeks post injection that were immediately fixed in 10% Neutral Buffered Formalin. The fixed tumors were embedded in paraffin and stained with hematoxylin and eosin. Images were collected and analyzed using the NanoZoomer Digital Pathology software (Hamamatsu).
6
Xenograft Tumor Formation from Human iPSCs
7
Histological Analysis of Whole Eyes
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Animals were euthanized by overdose of CO2 and their whole eyes removed. Tissues were fixed overnight (O/N) in 4% paraformaldehyde at room temperature, processed, and frozen or embedded in paraffin. Serial sections were cut at 5 μm (paraffin) or 15 μm (frozen) and either used for hematoxylin and eosin (H&E) staining or immunohistochemical analysis. Visualization and imaging were performed with a Nikon Tie-A1 confocal microscope (Nikon Instruments Inc., Melville, NY, USA) and NanoZoomer Digital Pathology software (Hamamatsu, Iwata City, Shizuoka Pref., Japan).
8
Adipose Tissue Morphometric Analysis
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Adipose tissue was dissected, fixed, and embedded in paraffin, sectioned and stained with H&E. Images were captured using a NanoZoomer slide scanner system with NanoZoomer Digital Pathology software (Hamamatsu). Images were analyzed and cell diameter measured using ImageJ software and presented as number of cells in each cell size category.
9
Immunohistochemical Analysis of Cervix Tissues
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All human cervical specimens were obtained from patients treated at Tokyo Medical University Hospital (Tokyo, Japan) under an approved Institutional Review Board protocol (T2020-0324). The human and murine uterine cervix specimens were fixed with formalin and embedded in paraffin. Sections were stained with Hematoxylin and Eosin or incubated with a primary antibody, and a second antibody. They were visualized using the EnVision DAB kit (Dako A/S, Denmark). Stained images were taken and analyzed by NanoZoomer Digital Pathology software (Hamamatsu Photonics, Japan). The specific information of antibodies is described in Supplemental Table S3 . Six mice in each group for scoring of IHC staining, IHC staining cells were counted from five locations randomly selected from the section of the uterine cervix of WAPL transgenic mice and human uterine cervix by ImageJ software. Either nucleus or cytoplasmic signal above background was scored as positive.
10
Human iPSC-Derived Tumor Xenografts
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Human iPSCs cultured in 6- well plates were dissociated with DPBS containing 0.5 mM EDTA and approximately 1 × 107 dissociated cells were collected and resuspended in 400 μl culture medium supplemented with 25 mM HEPES (pH7.4) and stored on ice. Then, 50% volume (200 μl) of cold Matrigel (Corning, 354277) was added and mixed with the cells. The mixture was injected subcutaneously into NSG mice (JAX No. 005557) at 150 μl per injection site. Visible tumors were removed 6–8 weeks post injection and were immediately fixed in 10% Neutral Buffered Formalin. The fixed tumors were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Images were collected using the NanoZoomer Digital Pathology Software (Hamamatsu).
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