Cells were seeded in Ibdi 2 well silicone inserts (Ibdi cat# 81176) in 24 well plates; 20,000 cells per insert well to obtain 95–100% confluency the next day. To inhibit further cell proliferation, the cells were treated with 50 μg/ml Mitomycin C (cat# M4287, Sigma) in complete culture medium for 2 h. The cells were washed twice with complete medium, and inserts were removed. The cell gaps were photographed (timepoint 0). Cells were returned to the tissue culture incubator and photographed again after 20, 48 and 72 h, at the same positions along the gap. The distance of cell migration was calculated by digitally drawing ten evenly spaced horizontal lines from edge to edge of the wounds at 0 and 20 h using NIS Elements BR 2.3 (Nikon). The mean value was calculated for each scratch. For each experiment, 4–10 scratches were measured per cell type. Three independent experiments were analyzed. To determine the statistical relevance of differences in migration between the different cell lines, Student’s t-test was performed.
Nis elements br 2
Manufactured by Nikon
Sourced in Japan
NIS-Elements BR 2.30 is a software package for microscope control, image acquisition, and analysis. It provides a comprehensive set of tools for managing and processing microscopy data.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Laminin, by Thermo Fisher Scientific (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Fura 2 acetomethoxy ester, by Thermo Fisher Scientific (2 mentions)
Lambda 10b shutter, by Sutter Instruments (2 mentions)
Mitomycin c, by Merck Group (1 mentions)
Coolpix 4500, by Nikon (1 mentions)
H550s microscope, by Nikon (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
Coolsnap es, by Nikon (1 mentions)
Eclipse t2000 u microscope, by Nikon (1 mentions)
Eclipse te2000 u inverted microscope, by Nikon (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
Dxm1200c digital camera, by Nikon (1 mentions)
Eclipse e400 microscope, by Nikon (1 mentions)
Ab2605, by Abcam (1 mentions)
Haematoxylin qs, by Vector Laboratories (1 mentions)
E6000 eclipse microscope, by Nikon (1 mentions)
19 protocols using nis elements br 2
1
Cell Migration Assay Using Wound Healing
2
Intestinal Histomorphology and Goblet Cell Analysis
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The histomorphology and the count of goblet cells in the jejunum were determined according to our previous study [30 (link)]. Briefly, each tissue sample was used to prepare five slides and each slide had three sections (5 μm thickness), which were stained with hematoxylin and eosin for intestinal morphology analysis of 20 intact well-oriented crypt–villus units each section (Scion Image software, Version 4.02, 2004). Periodic Acid Schiff and Alcian Blue (PAS-AB) were used for counting goblet cells. The number of positively stained goblet cells was measured (NIS-Elements BR 2.3; Nikon France SAS), and the values obtained from 10 villi by each small-intestinal segment were averaged.
3
Quantifying Intracellular Legionella in Acanthamoeba
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Acanthamoeba co-cultures with L. pneumophila were analysed at different time points to determine the maximum possible number of L. pneumophila cells within an amoeba cell. This time point was considered the optimal time to apply the release methods. Briefly, 1 mL of the co-culture sample was washed twice by centrifugation at 1000 × g for 5 min by adding fresh phosphate-buffered saline (PBS). Then, 900 μL of the supernatant was discarded, and the pellet was resuspended in the remaining PBS. From this, 10 μL was placed onto a 10-well Teflon slide (Medco Health Solutions, Inc., Germany). Slides were incubated at 30 °C for 30 min to let the cells attach to the slide surface. The samples were fixed by incubation for 10 min at RT in 20 μL of 4 % paraformaldehyde (v/v PBS), washing once with PBS, and dehydrating for 3 min in an aqueous ethanol series (50, 80, and 96 %). Cells were then stained with the MONOFLUO™ Legionella pneumophila IFA Test Kit, which uses FITC-labelled monoclonal antibodies to detect the major outer membrane protein of L. pneumophila. Slides were then investigated using a Nikon Eclipse 8000 epifluorescence microscope, and photographs were processed with the software NIS Elements BR 2.3 (Nikon).
4
Histological Analysis of Calvarial Bone
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The excised calvarial specimens were prepared for non-demineralized histological analysis.
Each bone specimen was fixed in 10% neutral-buffered formalin, rinsed with water, dehydrated using a series of increasing concentrations of ethanol solutions, cleared with xylene, and then embedded in methyl methacrylate. After polymerization, the calvarial bone blocks were oriented to radiography to obtain cross-sections cut parallel to the sagittal plane.
Each such section was ground down to a thickness of 300 μm using the exact grinding system (Exakt Apparatebau GmbH Norderstedt, Germany), and polished down to 100 μm. Each section was stained with Stevenel's blue and Van Gienson's picro fuschin before visualization using standard light microscopy.
Images of histological sections were obtained using a light microscope (Olympus Bx 4500) connected to a digital camera (Nikon Coolpix 4500) equipped with a video and image analysis system (NIS Elements BR 2.3, Nikon France S.A.S., France). The images that corresponded to the three successive cross-sections of each defect center were analyzed. To overcome the problem arising from the similarity of bone and material pixels on the digitized and calibrated images, the newly formed bone area and residual particles were delineated manually. The total area of newly formed bone was calculated by adding the areas measured and reported in mm 2 .
Each bone specimen was fixed in 10% neutral-buffered formalin, rinsed with water, dehydrated using a series of increasing concentrations of ethanol solutions, cleared with xylene, and then embedded in methyl methacrylate. After polymerization, the calvarial bone blocks were oriented to radiography to obtain cross-sections cut parallel to the sagittal plane.
Each such section was ground down to a thickness of 300 μm using the exact grinding system (Exakt Apparatebau GmbH Norderstedt, Germany), and polished down to 100 μm. Each section was stained with Stevenel's blue and Van Gienson's picro fuschin before visualization using standard light microscopy.
Images of histological sections were obtained using a light microscope (Olympus Bx 4500) connected to a digital camera (Nikon Coolpix 4500) equipped with a video and image analysis system (NIS Elements BR 2.3, Nikon France S.A.S., France). The images that corresponded to the three successive cross-sections of each defect center were analyzed. To overcome the problem arising from the similarity of bone and material pixels on the digitized and calibrated images, the newly formed bone area and residual particles were delineated manually. The total area of newly formed bone was calculated by adding the areas measured and reported in mm 2 .
5
Systematic Sampling of Lumbar DRG
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For systematic sampling, two lumbar DRG (L4-L5) per mice were collected, fixed in PFA, cryoprotected (30% sucrose), frozen at −20°C and then were cut on a cryostat at 8 µm. Double-labeling with Neuron-specific β–III tubulin (clone TUJ-1, 1∶1000, R&D systems, Lille, France) and with SP or CGRP was performed. Each DRG section was photographed under fluorescence microscope (200×) in a systematic fashion. Immunoreactive DRG neurons were counted and only the area containing neurons was measured with NIS-Elements BR 2.30 software (Nikon). The density of TUJ-1(+) neurons was expressed as neurons/mm2. The density of peptidergic neurons was expressed as CGRP(+) or SP(+) neurons/TUJ-1(+) neurons.
6
DRG Neuron Calcium Imaging Protocol
7
Histochemical Analysis of Muscle Fiber Types
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Serial cross-sections were taken at 9 μm in a cryostat (CM 1900 LEICA, Instrument GmbH, Germany) at −20 °C and placed on glass slides for the histochemistry analysis. These serial sections were stained for myofibrillar adenosine triphosphatase (mATPase) following the acid (pH 4.3, pH 4.55 and pH 4.6) preincubations [28 ]. The optimum pHs of the preincubation solutions were searched carefully to visually distinguish at least three levels (light, medium and dark) of staining intensities. The histochemical activity of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), an enzyme frequently used as a marker for the oxidative capacity of myofibers, was also qualitatively estimated on 9-μm-thick sections [28 ]. Histochemical serial sections were visualised with a Nikon H550S microscope (Nikon, Metrology Europe NV, Leuven, Belgium) and image analyser software NIS-Elements Br 2.30. All sections were carefully surveyed to find two regions that were free of artefacts that contained between 200 and 250 fibres. With the use of the mATPase staining stage after acid preincubation and the NADH-TR staining stage, muscle fibres were identified. The histochemical fibre types were classified into four major types (I, IIA, IIB and IIX). Once each individual fibre was identified, the minor diameter was determined.
8
Immunohistochemical Analysis of Human Gastric Tissue
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Cryosections or a paraffin-embedded gastric tissue microarray7 (link) (kind gift from Dr Paul Harris, Pontifical Catholic University of Chile, Santiago, Chile) prepared from human gastric tissue were labeled with anti–HLA-DR (cryosections: L243, BD 340689; tissue array: LN-3, ab166777; Abcam, Cambridge, UK) to stain DCs and with anticytokeratin FITC (cryosections: CAM5.2, BD 347653; tissue array: C11; 4545; Cell Signaling Technology, Danvers, MA) to stain epithelial cells, as previously described.4 (link), 7 (link) Cell nuclei were stained with 4′,6-diamidino-2-phenylindole. Samples were imaged on a Nikon Eclipse T2000-U fluorescent microscope (Nikon, Melville, NY) equipped with a CoolSnap ES digital camera and NIS Elements BR2.30 (Nikon). ImageJ V1.4848 (link) was used to measure epithelial cell length and count DCs that were in direct contact with the epithelium.
9
Electrospinning of Collagen-PCL Nanofibers
10
Renal Tissue Histological Visualization
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The renal slices were stained with hematoxylin and eosin. The stained sections were then visualized using a Nikon Eclipse 80i microscope (Nikon Co), and the images were captured using the NIS-Elements BR 2.30 software (Nikon Co).
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