All immunostained sections were digitally imaged using a Nikon CoolScope (Nikon). Immunohistochemistry of mouse liver was performed as described previously78 (link) using (a) anti-phospho-IRE1α (Abcam), (b) anti-phospho-JNK (ThermoFisher Scientific), and (c) anti-cleaved lamin (small subunit, Cell Signaling Technology) antibodies and 3,3′-diaminobenzidine (DAB) as the chromogen. Phospho-IRE1α, phospho-JNK, and cleaved lamin A indices were calculated by dividing the DAB-positive area by the region of interest (ROI), and results are expressed as A.U. The cleavage of lamin is a well-characterized event in apoptosis79 (link). TUNEL staining was performed as previously described17 (link), 22 (link) using the FragELTM DNA Fragmentation Detection Kit (EMD Millipore-Calbiochem) in accordance with the manufacturer’s instructions. TUNEL-positive cells within 0.145 mm2 of the ROI were counted, and TUNEL indices were calculated as the number of TUNEL-positive cells per unit area (in mm2); results are expressed as A.U.
Coolscope
Manufactured by Nikon
Sourced in Japan, United States
The Coolscope is a compact and versatile digital microscope designed for laboratory applications. It features a high-resolution camera and advanced optics that enable users to capture detailed images and videos of microscopic samples. The Coolscope's core function is to provide a reliable and efficient tool for visualization, analysis, and documentation of various specimens in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Sp5 laser scanning confocal microscope, by Leica (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Anti phospho ire1α, by Abcam (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Tgf β3, by Thermo Fisher Scientific (1 mentions)
Its premix, by BD (1 mentions)
Eclipsenet, by Nikon (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
Rankl, by American Type Culture Collection (1 mentions)
Nis elements software, by Nikon (1 mentions)
Gt 9800f, by Epson (1 mentions)
H 7100, by Hitachi (1 mentions)
Epon 812, by TAAB (1 mentions)
Ki 67 antibody, by Agilent Technologies (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Agilent Technologies (1 mentions)
Anti rat cd68 antibody, by Bio-Rad (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Vt1200, by Leica (1 mentions)
24 protocols using coolscope
1
Quantitative Analysis of Apoptosis Markers
2
Histological Examination of Skin Tissues
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Skin tissues fixed in 4% buffered paraformaldehyde were paraffinized and
sectioned into 5-µm-thick sections. The sections were stained with hematoxylin
and eosin (H&E) and examined under a Nikon COOLSCOPE (Nikon Instruments,
Inc., NY, USA).
sectioned into 5-µm-thick sections. The sections were stained with hematoxylin
and eosin (H&E) and examined under a Nikon COOLSCOPE (Nikon Instruments,
Inc., NY, USA).
3
Evaluating Tumor Angiogenesis and Lymphatics
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Images of positively stained vessels were captured using a digital light microscope (Nikon Coolscope, Nikon Corporation, Japan) between 10x and 400x magnification. The images of tumor fields were captured to be representative of the entire tumor, using a raster pattern which allowed for captured fields to be random and avoid overlap. Between 20 and 30 fields per tumor were assessed. The images were analyzed using Image-Pro Plus (Version 5, Media Cybernetics, Perth Australia).
Vessels were assessed as the number of positively stained vessels per tumor area to provide a microvascular density index.
The lymphatic vessel density (LVD) assessments were performed by researchers blinded to the patient outcome data.
Vessels were assessed as the number of positively stained vessels per tumor area to provide a microvascular density index.
The lymphatic vessel density (LVD) assessments were performed by researchers blinded to the patient outcome data.
4
Chondrogenic Differentiation of MSCs
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For chondrogenic differentiation, cells were grown into a pellet in 15 ml tubes, at approximately 250,000 cells/tube, in culture medium (control cells) or in chondrogenic medium for 7 weeks. rMSCs’ chondrogenic medium consisted of culture medium supplemented with 15% FBS, 1 mM piruvate, 100 nM dexamethasone, 37.5 μg/ml ascorbic-2-phosphate acid and 10 ng/ml Transforming Growth Factor-β3 (TGF-β3, Peprotech, Rocky Hill, NJ, USA). hMSCs’ “chondrogenic medium” consisted of 4.5 g/L glucose culture medium serum-free and supplemented with ITS+premix (1:100, Becton Dickinson, Franklin Lakes, NJ USA), 1 mM piruvate, 100 nM dexamethasone, 50 μg/ml ascorbic acid 2-phosphate, 10 ng/ml TGF-β3. Chondrogenic differentiation was evaluated by Safranin O staining. hMSC and rMSC pellets, treated with chondrogenic medium or with culture medium only, were fixed with 10% neutral buffered formalin for 30 minutes, paraffin embedded by standard methods and cut into 7 μm sections. Sections were stained with Hematoxylin-eosin and Safranin O 0.1%. The slides were mounted and photos were taken using a Nikon Coolscope instrument (Nikon Instruments S.p.A., Italy). Safranin O is a histological cartilage-specific staining that binds to proteoglycans and glycosaminoglycans resulting in a red/orange staining.
5
Immunostaining and Quantification of Amyloid-Beta Plaques
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Polyphenol-treated and control mice were sacrificed by CO2 euthanasia after the ten day treatment. Brain tissues were processed and stained by immunohistochemistry as described previously[9 (link)], with antibodies against β-amyloid (1:100; Abcam, Cambridge, UK) for Aβ deposits.
After immunostaining, tissue sections were examined by light microscopy (Nikon Cool scope, Nikon, Düsseldorf, Germany). Aβ plaques in cortex and hippocampus were counted by a certain diameter and clear deposition for plaques. Then, the percentages of areas with specific immune-reactivity (IR) in the cortex and hippocampus were analyzed using the software Meta Morph Offline 7.1 (Molecular Devices, Toronto, Canada).
After immunostaining, tissue sections were examined by light microscopy (Nikon Cool scope, Nikon, Düsseldorf, Germany). Aβ plaques in cortex and hippocampus were counted by a certain diameter and clear deposition for plaques. Then, the percentages of areas with specific immune-reactivity (IR) in the cortex and hippocampus were analyzed using the software Meta Morph Offline 7.1 (Molecular Devices, Toronto, Canada).
6
Osteoclastogenesis Quantification Assay
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RAW 264.7 cells (American Type Culture Collection [ATCC], Manassas, VA, USA), a murine monocytic cell line that differentiates in the presence of RANKL, was used for measurements of osteoclastogenesis and osteoclast activity. Cells were amplified on nonadherent culture substratum in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum (FBS) and penicillin-streptomycin in a 37 °C, 5 % CO2, humidified incubator and were passaged by manual dissociation. Cells were seeded on 16-well osteologic slides (BD BioCoat; BD Biosciences Discovery Labware, Billerica, MA, USA) at 0.125 × 104 cells/0.25 ml/well in triplicate and were allowed to adhere for 24 h, then stimulated to differentiate (30 mg/ml RANKL; PeproTech, Rocky Hill, NJ, USA) with or without drug for 7 days. Plates were subsequently rinsed, bleached, dried, and analyzed for matrix degradation using a Nikon COOLSCOPE (Nikon Instruments, Melville, NY, USA). Matrix resorption was quantified using EclipseNet (Nikon Instruments)/Visiopharm (Visiopharm, Hørsholm, Denmark) software (see Additional file 1 ).
7
Quantifying Aortic Fat Deposition
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After sacrifice, aortas were dissected from heart to iliac branching, with particular attention at removing external fat in order to stain exclusively sub-intimal aortic fat [14] . Aortic lesion stainings were performed as previously described [29] ; images were acquired using a Nikon Coolscope (Nikon), and fat deposits were quantified with NIS elements software (Nikon). For histological analysis, aortas were formalin-fixed and paraffin-embedded. Representative sections of each specimen were stained with hematoxylineosin and were examined by a pathologist to evaluate atherosclerotic lesions.
8
Confocal Microscopy and Image Analysis
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Immunofluorescence images were acquired using a Leica SP5 laser scanning confocal microscope and a 40 × oil objective with pinhole set to 1 AU and line average set to 3, and analysed using Volocity software; background thresholds were set based on young adenoviral control samples and antibody control samples. Immunohistochemistry images were acquired using a Nikon CoolScope at 10 or 20 × magnification and analysed using ImageJ software. HMGB1 immunofluorescence images were quantified using CellProfiler, an open-access image analysis programme ( www.cellprofiler.org ).
9
Multimodal Imaging Analysis Protocol
10
Multi-Tissue Ultrastructural Analysis
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Dahl rats were anesthetized by intraperitoneal injection of chloral hydrate (0.35/Kg body weight) and fixed by intracardiac perfusion with 1% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. After rinsing with phosphate buffered saline (PBS), pH 7.4, slices of fixed tissues, obtained from brain, spinal cord, heart, kidney and liver, were postfixed with 1% OsO4 for 4 hr at 4 °C, treated with 1% uranyl acetate for 1 hr at 4 °C, dehydrated in ethanols, and embedded in Epon812 (TAAB Laboratories Equipment Ltd, Berks, UK). For light microscopy, 1 μm thick sections were cut and stained with toluidine blue. Photographs of various tissues were taken using a digital microscope (COOL SCOPE, Nikon Instruments, Inc., Tokyo, Japan). For electron microscopy, thin sections, stained with uranyl acetate and lead citrate, were examined with a Hitachi H-300 or H-7100 electron microscope. Electron micrographs were produced with a film scanner (GT-9800F, Epson, Tokyo, Japan), and both light and electron microscopic images were compiled using Adobe Photoshop Element software.
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