For quantification of RGCs, eyeballs were cryosectioned at a thickness of 20 μm. The sections were stained for RBPMS (a selective marker of ganglion cells in the mammalian retina) to identify RGCs. Fluorescence images were acquired with a 10x objective using a fluorescence microscope (VS120, Olympus, Japan). At least three sections near the maximum diameter of the eyeball per retina were quantified. In intact control retinas, 300 to 500 RBPMS+ cells were generally counted per section. The cell count was normalized to the length of the ganglion cell layer (measured using OlyVIA software for every section), and the average value for each retina was recorded for the subsequent statistical analysis. For quantification of RGCs in whole-mount retinas, fixed whole-mount retinas were first stained for Tuj1, radially cut into a petal shape and viewed under a fluorescence microscope (VS120, Olympus, Japan, 10X objective). Generally, six to eight fields (each 0.4 mm x 0.4 mm) of each retina were used for RGC quantification, and then the values were averaged to obtain one value for each retina for the subsequent statistical analysis.
Vs120
Manufactured by Olympus
Sourced in Japan, Germany, United States
The VS120 is a virtual slide system designed for the digitization and analysis of histological samples. It captures high-resolution images of entire slides and enables remote viewing and sharing of digital slides.
Automatically generated - may contain errors
Lab products found in correlation
Fv1200, by Olympus (5 mentions)
Fv3000, by Olympus (4 mentions)
Cm3050, by Leica (4 mentions)
Cryostat, by Leica (4 mentions)
H 1200, by Vector Laboratories (3 mentions)
Tcs sp8, by Leica (3 mentions)
Permount, by Thermo Fisher Scientific (3 mentions)
Vt1200, by Leica (3 mentions)
Image pro plus 6, by Media Cybernetics (3 mentions)
Olyvia software, by Olympus (3 mentions)
Cellsens dimension, by Olympus (3 mentions)
Fc receptor blocking, by Vector Laboratories (2 mentions)
Vt1000s, by Vector Laboratories (2 mentions)
Fluoview fv1200, by Leica (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Cm1900, by Leica (2 mentions)
Prolong diamond antifade reagent, by Thermo Fisher Scientific (2 mentions)
Neurotrace 640 660, by Thermo Fisher Scientific (2 mentions)
A1 32 edge 5mm 100 177, by NeuroNexus (2 mentions)
Alexa 568, by Thermo Fisher Scientific (2 mentions)
236 protocols using vs120
1
Quantification of Retinal Ganglion Cells
2
Quantifying Liver Fibrosis and Lipid Deposition
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Masson’s trichrome staining was used to measure fibrosis. Liver biopsies from the left anterior lobe were immediately fixed 10% neutral buffered formalin (Thermo Fisher Scientific) for 24 hour and subsequently stored in ethanol (70%). Immediately prior to staining, biopsies were embedded in paraffin, and transverse cross sections (5 μm) were subsequently cut and processed by the University of Toledo College of Medicine and Life Sciences Histology Core following standard staining procedures. Stained cross sections were viewed with a light microscope (Olympus VS120) using a ×20 objective. Four different cross sections of each biopsy were examined for fibrosis, as indicated by the abundance of blue collagen staining. Images were analyzed using ImageJ.
Oil Red O staining was used to measure lipid droplet accumulation. Liver biopsies from the left anterior lobe were immediately embedded in tissue freezing medium and frozen in liquid nitrogen. Transverse cross sections (8 μm) were processed by the University of Toledo College of Medicine and Life Sciences Histology Core following standard staining procedures. Stained cross sections were viewed with a light microscope (Olympus VS120) using a ×20 objective. Four different cross sections of each biopsy were examined for the abundance and size of red lipid droplets. Images were analyzed using ImageJ.
Oil Red O staining was used to measure lipid droplet accumulation. Liver biopsies from the left anterior lobe were immediately embedded in tissue freezing medium and frozen in liquid nitrogen. Transverse cross sections (8 μm) were processed by the University of Toledo College of Medicine and Life Sciences Histology Core following standard staining procedures. Stained cross sections were viewed with a light microscope (Olympus VS120) using a ×20 objective. Four different cross sections of each biopsy were examined for the abundance and size of red lipid droplets. Images were analyzed using ImageJ.
3
Cryopreservation and Lipid Quantification of Skeletal Muscle
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Muscles were cryopreserved by incubation with 2-methylbutane in a steel beaker using liquid nitrogen for 30 s, cryoembedded, and cryosectioned at 8 μm thickness. Tissue sections were stained following standard H&E protocol. Photomicrographs of skeletal muscle fiber were imaged under brightfield (VS120, Olympus). Muscle slides fixed in 3.7% formaldehyde were stained with 0.3% Oil Red O (in 36% triethyl phosphate) for 30 min. Images were taken in brightfield (VS120, Olympus). The relative concentration of lipid was determined by extracting the Oil Red O with isopropanol in equally sized muscle sections and quantifying the OD500 (optical density at 500 nm) in a 96-well plate.
4
Immunofluorescence Imaging of HEK293 Cells
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Transverse brain sections of 12-μm thickness were prepared and HEK293 cells were seeded on LAB-TEK chamber slides (Thermo Fisher Scientific, 154453) for immunoflorescence assays as previously described (18 (link), 42 (link)). The following antibodies were used: anti-RNF220 (1:200; Sigma-Aldrich, HPA027578), anti-PDGFRα (1:400; R&D, AF1026), anti-CC1 (1:200; Millipore, OP80), anti-Sox10 (1:300; Oasis Biofarm, OB-PGP001), anti-BrdU (1:200; Bio-Rad, MCA6144), anti-Ki67 (1:200; Abcam, ab15580), and fluorescence-conjugated secondary antibodies, Alexa Fluor 488/555/594 donkey/goat anti-rabbit/mouse/rat/ guinea pig immunoglobulin G (1:400; Invitrogen; A11076, A11055, A21202, A48269, A21206, A31572). Images were captured using a light microscope (Olympus, VS120) and analyzed with ImageJsoftware.
For myelination analysis, 20-μm brain coronal sections were prepared with a cryostat microtome (Leica, CM1850UV) and stained with the Black-Gold II kit (Biosensis, TR-100-BG) according to the manufacturers’ instructions. Images were captured using a light microscope (Olympus, VS120) and analyzed with ImageJ software.
For myelination analysis, 20-μm brain coronal sections were prepared with a cryostat microtome (Leica, CM1850UV) and stained with the Black-Gold II kit (Biosensis, TR-100-BG) according to the manufacturers’ instructions. Images were captured using a light microscope (Olympus, VS120) and analyzed with ImageJ software.
5
Histological Analysis of Murine Lungs
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Mice lung tissue samples were fixed in 10% formalin, paraffin-embedded, and stained with standard hematoxylin and eosin in 3-μm sections. The tissue slides were scanned on a VS120 Olympus with high resolution to perform histological analysis of parenchyma area.
6
Multimodal tissue imaging of ACE2, neurons, and blood vessels
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Brain sections were stained for neurons (NeuN) and blood vessels (collagen IV), and kidney sections for ACE2. The tissue was blocked with 5% donkey serum for 1 h at RT and the primary antibodies incubated overnight. The primary antibodies were mouse anti-NeuN (1 : 200, Cat# MAB377, Millipore Sigma, Billerica, MA, USA), anti-collagen IV (1 : 200, Cat# SAB4500369, Millipore), and anti-ACE2 antibody (1 : 100; Life Technologies. Cat# MA532307). The secondary antibodies were donkey anti-rabbit-Cy5 for collagen IV and ACE2, and donkey anti-mouse-Cy5 for the NeuN (Jackson ImmunoResearch, Inc., West Grove, PA, 1 : 500), which were added and incubated for 1.5 h at RT. Immunofluorescence was visualized by using an epifluorescence microscope (VS120 Olympus).
7
Quantifying Phagocytic Microglia in White Matter Lesions
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Lesion areas were selected on the basis of the characteristics described below using LFB/PAS staining and immunohistochemistry for Ki‐M1P and myelin proteins (MBP, MAG, MOG, CNPase, and PLP). Borders of specific lesion areas were outlined on sections where IHC for microglia/macrophage markers had been performed, and cell densities were determined using a 10 × 10 ocular morphometric grid at a 400× magnification (Olympus, Japan). Comparable regions of normal white matter were analyzed in age‐matched controls. Results are expressed as cells/mm2. To determine ongoing phagocytosis, Iba1 or Ki‐M1P and MBP‐double‐labeled sections were scanned for intracytoplasmic myelin fragments. Co‐expression of marker proteins on phagocytes was investigated on sections double‐labeled for Tmem119 or P2ry12 and Iba1. Fluorescently‐labeled sections were scanned using the virtual slide microscope VS 120 and visualized using the VS‐ASW software (at 200× magnification) (Olympus, Tokyo, Germany). Immunopositive cells were counted manually using Image J (v. 1.51w NIH, USA) to determine the percentage of Tmem119/Iba1 and P2ry12/Iba1 double‐positive cells.
8
Virus Amplification and Histological Analysis
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For virus amplification, a moderate amount of HSV-1 was incubated with Vero cells for 72 hours, all culture supernatants were aspirated, and adherent cells were scraped down as collection. The viral suspension was then obtained after repeated freezing and thawing three times, and the plaque assay was performed by the standard plaque assay to measure the titer of the virus. Lung tissues from the HSV-1–infected and uninfected mice were fixed with 10% phosphate-buffered formalin, embedded into paraffin, sectioned, stained with H&E solution, and examined by microscopy (OLYMPUS VS120) for histological analysis.
9
Mandible Decalcification and Histological Analysis
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The mandible samples (n = 5) were decalcified in 10% (w/v) EDTA at 37 °C and a pH of 7.4 for one month in a multifunctional microwave tissue processor (Milestone Medical, Kalamazoo, MI, USA) with weekly changes of the solution prior to embedding in paraffin. Consecutive serial sections (5 µm) were subsequently deparaffinised and stained with Masson’s Trichome (TRI Histology facility with Tissue-Tek automated stainer and coverslipper). The stained slides were scanned using a 40× objective lens on a virtual slide microscope (VS120; Olympus, Tokyo, Japan) for subsequent morphological analysis.
10
Digital Pathology Cervical Cell Categorization
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TCT specimens were digitized to images using a microscope with a 40× objective lens (model: VS120, Brand: Olympus) and cut into 2048×2048 pixels for labeling (Fig. 1 ). Two clinical pathologists independently labelled the squamous epithelial cells with bounding boxes as NILM, ASC-US, LSIL, ASC-H, HSIL, and SCC using LabelImg software, which is a graphical image annotation tool (https://github.com/tzutalin/labelImg ), according to the Bethesda reporting system. After labeling, all data were split into single-cell images according to the labeled coordinates of bounding boxes and rescaled to 224×224 pixels according to the shortest side (Fig. 1 ). To reduce the inter- and intra-observer variability, an annotated review was conducted of the original pixel images of the single cells one month following the initial labeling.
In order to prevent the problem of over-fitting, where the model may not accurately predict additional data, data augmentation on the training set using image flipping was performed. Data normalization was completed to ease the redundant image differences caused by the different environments and staining workflow of multiple hospitals (18 (link)).
In order to prevent the problem of over-fitting, where the model may not accurately predict additional data, data augmentation on the training set using image flipping was performed. Data normalization was completed to ease the redundant image differences caused by the different environments and staining workflow of multiple hospitals (18 (link)).
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