Tissues were fixed in formalin, embedded in paraffin, and sectioned (5μm). After deparaffination, sections were stained for CD31 (SZ31; Dianova DIA-310) 1:10, and goat anti rat IgG-HRP (eBioscience 18-4818) 1:50. The capillary area was determined by analyzing CD31-positive area using NIS Elements BR image analysis. In addition, the number of capillaries were counted per view of field (x100) (control n=9; VB-111 n=6).
Nis elements br
Manufactured by National Instruments
NIS Elements BR is a software platform for microscopy imaging and analysis. It provides a comprehensive set of tools for image acquisition, processing, and analysis across a wide range of microscopy techniques.
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Lab products found in correlation
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Ds ri2 camera, by National Instruments (1 mentions)
Eclipse e600, by Nikon (1 mentions)
B 41 microscope, by Olympus (1 mentions)
Methyl salicylate, by Merck Group (1 mentions)
Methyl salicylate, by Nikon (1 mentions)
Cm dii, by Thermo Fisher Scientific (1 mentions)
Eclipse ti, by Nikon (1 mentions)
10 protocols using nis elements br
1
Quantifying Capillary Area and Density
2
Histologic Analysis of Humerus Samples
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For histologic analysis, we chose the associated humerus. Before sectioning, photography, photogrammetry, and CT scanning of the humerus were conducted. Molding and casting were made from the specimen for later research. The mid-shaft of the humerus was sampled for the examination. The sample was prepared and transversely thin sectioned following Lamm (2013) . The slides were examined using Nikon Eclipse E600 POL petrographic polarizing microscope with a lambda 530 nm plate. The bone cross-section was photographed using a combination of Nikon DS-Ri2 camera and NIS-Elements BR (ver. 4.13) software. Adobe Photoshop (ver. 21.2) is used for image enhancement and tracing LAGs. Bone wall thickness and area of the transverse section were quantified by Image J (ver. 1.53; Schneider, Rasband & Eliceiri, 2012 (link)).
3
Histomorphological Analysis of Mesenteric Arteries
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Third-order mesenteric arteries were fixed in 10% buffered formalin and embedded in paraffin in a routine manner. Four µm thick sections were cut using a Leica 2025 (Leica Biosystems Inc., Buffalo Grove, IL, USA) rotating microtome and stained by hematoxylin and eosin (H+E). Histomorphological analysis of mesenteric arteries slides was performed using an Olympus B×41 microscope (Olympus Corporation, Tokyo, Japan). Digital images were processed using NIS Elements BR software (NIS-Elements BR 3.2 64-bit NIS AR 3.0) which was used to calculate the width of the media. The measurement of the thickness of the middle layer of the mesenteric arteries was made at a uniform 200× magnification. Five sections from each specimen were measured [59 (link)].
4
Membrane Hole Size Analysis
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To identify membrane holes, the threshold value was set by using the signal/noise ratio option in NIS-Elements BR software. The setting used was the result of subtracting 6 of standard deviations from average fluorescence intensity of the bilayer areas between holes. This threshold was automatically applied for analysis of the areas and the sizes of membrane holes.
5
Embryological Observation of Flower Development
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For embryological processes observation, 23 flowers (KAM, KON, PIL, RUR) were cleared using methyl salicylate method, and divided into two groups. I. Flowers at preanthesis and anthesis stages were cleared in a mixture of 100% ethanol and methyl salicylate (Sigma-Aldrich) in proportions 3:1; 1:1; 1:3, 0:3 two hours each change. II. At postanthesis stages prior to clearing, pre-softening of the tissues using Schiff reagent was applied. Cleared pistils and/or anthers were transferred onto Ray chambers in a drop of methyl salicylate and observed under Nikon Eclipse Ni light microscope with Nomarski differential interference contrast (DIC) optics, equipped with camera Nikon DS-Filc and NIS-Elements BR Viewer imaging software ver. 4.10 (57 (link), slightly modified). For the study, 10 randomly selected ovules from each flower were analyzed.
6
Microscopic Analysis of Titanium-Polymer Bonds
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Titanium alloy–polymer bonds were subjected to microscopic observations. The bonds were checked before detachment and the surface of the polymer sample was observed after detachment. The tests were performed using a Nikon Eclipse MA200 light microscope. The observations were made at magnifications in the range of 100–500x. The images were recorded with a Visitron Systems digital camera with Spot Advanced and NIS Elements BR software. Metallographic specimens were prepared using a grinding and mechanical polishing process.
Microscopic observations were also carried out using a Phenom XL scanning electron microscope at magnifications of 500–2000x. An accelerating voltage range of 5–25 kV was applied during the experiments. Material contrast observations were performed using SE and BSE detectors.
Microscopic observations were also carried out using a Phenom XL scanning electron microscope at magnifications of 500–2000x. An accelerating voltage range of 5–25 kV was applied during the experiments. Material contrast observations were performed using SE and BSE detectors.
7
Immunofluorescence Analysis of Splenic Immune Cells
8
Cell Labeling and Imaging in Embryos
9
Lung Structural Analysis Post-μCT Imaging
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To analyze changes in lung structure post‐µCT imaging, lungs were placed in 70% ethanol and processed for paraffin embedding. 5 µm thick paraffin sections were stained with standard hematoxylin‐eosin or trichrome visualize fibrosis. Microfil was visible as a grainy brown substance in some vasculature but did not withstand processing. Images were taken on a Nikon Eclipse DS‐Fi3 microscope using NIS Elements BR software (v 5.30.03).
10
Cryosectioning Mouse Tibialis Anterior
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Harvested mouse TA muscles were immediately weighed and fixed in 4% (w/v) paraformaldehyde for 16 h at 4˚C and then placed in 30% sucrose for overnight incubation. The TA muscles were then embedded in Tissue Freezing Medium (Triangle Biomedical Sciences), and a Thermo HM525 cryostat was used to prepare 10 μm sections from the muscle mid-belly. All sections were examined and photographed using a Nikon Eclipse Ti automated inverted microscope equipped with NIS-Elements BR digital imaging software.
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