Sections were analyzed by use of an Olympus BX50 fluorescence microscope (mercury source) equipped with a BX-FLA reflected light attachment and vertical illuminator. Lumogallion fluorescence was acquired using a U-MNIB3 filter cube (bandpass λex: 470–495 nm, dichromatic mirror: 505 nm, longpass λem: 510 nm) and ThS fluorescence by use of a U-MWBV2 filter cube (bandpass λex: 400–440 nm, dichromatic mirror: 455 nm, longpass λem: 475 nm, both from Olympus, UK). Images were captured using a ColorView III CCD camera using the CellD software suite (Olympus, SiS Imaging Solutions, GmbH). Light transmission values and exposure settings were fixed across respective treatments. Merging of fluorescence channels was performed using Photoshop (Adobe Systems Inc., US).
Color view 3
Manufactured by Olympus
Sourced in Germany, Japan
The Color View III is a compact digital camera system designed for microscopy applications. It captures high-quality images and videos of microscopic specimens, providing a detailed visual representation for analysis and documentation purposes.
Automatically generated - may contain errors
Lab products found in correlation
Cell d, by Olympus (3 mentions)
Celld software suite, by Olympus (2 mentions)
Bx50 fluorescence microscope, by Olympus (2 mentions)
Toluidine blue, by Merck Group (2 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Eclipse e600, by Olympus (1 mentions)
Goat anti rabbit igg conjugated to alexa 488, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse igg conjugated to cy3, by Jackson ImmunoResearch (1 mentions)
Mesab, by Merck Group (1 mentions)
Mvx10, by Olympus (1 mentions)
Ckx41sf, by Olympus (1 mentions)
Cell f software, by Olympus (1 mentions)
Szx16, by Olympus (1 mentions)
Bx51 light microscope, by Olympus (1 mentions)
Rat anti mbp, by Merck Group (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
U ant transmitted light analyzer, by Olympus (1 mentions)
U mwbv2 cube, by Olympus (1 mentions)
U mnib3 fluorescence filter cube, by Olympus (1 mentions)
18 protocols using color view 3
1
Fluorescence Microscopy Protocol for Lumogallion and ThS
2
Histological Analysis of Femoral Condyles
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After MR scanning, the femoral condyles were thoroughly irrigated with normal saline and stored at −20° Celsius until further assessments were carried out. The histological sample processing was performed based on a protocol for non-decalcified sectioning.36 (link) Briefly, the specimens were dehydrated in an ascending alcohol series and embedded in Methyl methacrylate (Technovit 9100 new, Hereaus Kulzer, Weinheim, Germany). Sagittal sections were cut along the long axis of the Ethipins, generating 12–22 serial sections of each femoral condyle (section thickness: ∼200 µm) using a diamond edge bandsaw blade (Exakt, Nordenstedt, Germany). These sections were then ground to the final section thickness of ∼50 µm and polished with a plate grinder (Exakt microparallel-grinding System). Staining was performed with toluidine blue (0.1% toluidine blue in 0.1% sodium tetraborate; Merck, Darmstadt, Germany). For documentation and subsequent digital imaging, a binocular light microscope (Olympus BX50, Olympus, Hamburg, Germany), a colour CCD camera (Color View III, Olympus) and an imaging and documentation life science system (cell^D, Olympus) were used.
3
Immunohistochemical Staining of Extracellular Matrix Proteins
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Samples were fixed in 4% paraformaldehyde and embedded in paraffin before being cut and mounted on polysine glass slides. Those sections were deparaffinized and rehydrated using standard laboratory procedures, then post fixed in 4% paraformaldehyde in phosphate buffered saline for 10 minutes. Heat mediated antigen retrieval was carried out for 20 min in 0.05% citraconic anhydride buffer pH 7.4 for all stainings except for BMP-1 where 10 mM citrate buffer pH 6.0 was used. Then, primary antibodies in 1.25% bovine serum albumin (BSA) were applied (according to Table 2 ) and incubated overnight at 4°C. Negative controls were made omitting primary antibody, while for ADAMTS-2, ADAMTS-3 and BMP-1, isotype controls were used. After rinsing, secondary antibodies in 12.5% BSA were applied at room temperature for 1.5 hours. The secondary antibodies used were goat anti-rabbit IgG conjugated to Alexa 488 (Invitrogen, Carsbad, CA) and donkey anti-mouse IgG conjugated to Cy3 (Jackson Immuno Research Europe, Newmarket, UK), used at 5.0 and 1.4 µg/ml, respectively. The stained sections were mounted with prolong gold antifade (Invitrogen), containing DAPI for nuclear stain. Imaging was done using an upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III camera.
4
Anesthetizing and Imaging Zebrafish Larvae
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To evaluate morphological changes, larvae were anesthetized with 200 mg/ml 3-aminobenzoic acid methyl ester (MESAB, Sigma-Aldrich), mounted in 1.5% low melting temperature agarose in E3 medium and imaged using a stereomicroscope (Olympus MVX10) equipped with a color camera (ColorViewIII, Soft imaging System, Olympus).
5
Pt-NPPVP Cytotoxicity on HEI-OC1 Cells
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The HEI-OC1 cell line was kindly provided by Michael Morgan (Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany). Prior to Pt-NPPVP application 4000 cells were seeded in 96-well plates each containing 100 μl of the cell culture medium (high glucose DMEM and 10% FCS) and pre-cultured under permissive conditions (33°C, 10% CO2) for 24 h. As follows the cell culture medium was exchanged with those containing Pt-NPPVP concentrations between 50 μg/ml and 150 μg/ml and the cells were cultivated for further 48 h. For statistical assessment at least N = 6 experiments were considered and each cell culture assay was prepared in triplicates. To enable relative quantification of samples cultivated in different Pt-NPPVP concentrations HEI-OC1 cells without any treatment were used as reference, additionally cells exposed to 15% DMSO for 1.5 h were included to the study as negative control. Light microscopy (Olympus CKX41SF, Hamburg, Germany) was used to monitor changings in density and the morphology of the HEI-OC1 cells and images were taken digitally by the CCD colour camera (Olympus Color View III, Hamburg, Germany).
6
Morphological Changes in BALB/3T3 Fibroblasts
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Morphology of BALB/3T3 fibroblasts was observed at 1, 3, 6, 12 and 24 h of cell seeding on control or graphene-coated microscopic slides. Images were captured with an inverted microscope (Olympus IX71) equipped with Color View III cooled CCD camera and Cell^F software (Soft Imaging System) (Olympus, Tokyo, Japan).
7
Embryo Development Microscopy Protocol
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The morphological changes during development of both embryo types were documented using a stereo microscope (SZX 16 Olympus) equipped with a digital camera (Colorview III, Olympus, Germany) controlled by software Cell^D (Olympus, Germany). Images of one focal plane with adjusted contrast and brightness were assembled using Adobe Photoshop CS6. All data shown in this study were collected from at least six independent replicates.
8
Avian Hypothalamic Neuromorphometry
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Leghorn and Green-legged Partridge hen brains were collected, embedded in paraffin blocks, and cut into 10-µm thick frontal sections in a microtome. The slides were stained with cresyl violet. The ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), and anterior hypothalamus (AH) were analysed and photographed under the Olympus BX51 light microscope (Olympus, Tokyo, Japan) with a digital camera (Olympus Color View III). Morphometric assessment was conducted on randomly selected 20 microphotographs from every animal and every studied area. Neurons were counted in a grid of 6.25 × 10–2 mm2. Data were presented as means with standard deviation.
9
Assessing Spinal Cord Demyelination and Microglial Activation
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Ionized calcium-binding adapter molecule 1 (Iba1) and myelin basic protein (MBP) immunohistochemistry was performed to visualize respectively microglial cells and the myelin status of the spinal cord. Twenty micrometers of frozen sections were permeabilized in 1% Triton X/PBS for 5 min and incubated with the primary antibodies rat anti-MBP (1:500, Merck Millipore, Darmstadt, Germany) or rabbit anti-Iba1 (1:500, Wako, Neuss, Germany) at 4 °C overnight. Cy3-conjugated goat anti-rat or goat anti-rabbit (1:500, Merck Millipore, Darmstadt, Germany) were used as secondary antibodies, and cell nuclei were stained by DAPI (Invitrogen, Carlsbad, USA). After immunostaining, images were acquired with a fluorescence microscope (BX51; Olympus, Tokyo, Japan) equipped with a digital camera (Colorview III, Olympus, Tokyo, Japan). Demyelination (MBP score) was assessed blindly using the following scoring system: 1, no demyelination; 2, rare foci; 3, few areas of demyelination; and 4, confluent areas of demyelination. Microglial activation (Iba1 score) was evaluated blindly using the following system: 1, no activation; 2, mild activation; 3, strong activation; and 4, massive activation.
10
Cellulose Nanofibrils Optical Microscopy
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An Olympus BH-2 KPA microscope (Olympus Optical Co., Ltd, Japan) equipped with a high-resolution CCD colour camera (Olympus ColorView III) was used to observe the cellulose nanofibril samples, at different pH values and a consistency of 0.09 %. Samples were kept between cover slips and illuminated with linearly polarised light and analysed through a crossed polarizer. Images were captured and analysed using the analySIS software (Soft Imaging System GmbH).
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