Image acquisition was performed using a ×100 objective (NA 1.40) on a Leica (Nussloch, Germany) DM6000 upright epifluorescence microscope with a 12-bit cooled CCD camera (Micromax, Roper Scientific) run by MetaMorph software (Roper Scientific, Evry, France). Quantification was performed using MetaMorph software (Roper Scientific). Image folders were randomized before analysis. For morphological spine analysis exposure time was adjusted for each eGFP image to obtain best fluorescence to noise ratio and to avoid pixel saturation. For each neuron a well-focused dendrite was chosen, spine heads were manually delimited and their area were quantified. To assess KCC2-Flag clusters, exposure time was fixed at a non-saturating level and kept unchanged between cells and conditions. For cluster analysis, images were first flatten background filtered (kernel size, 3 × 3 × 2) to enhance cluster outlines, and a user defined intensity threshold was applied to select clusters and avoid their coalescence. Clusters were outlined and the corresponding regions were transferred onto raw images to determine the mean KCC2–Flag cluster number, area and fluorescence intensity. The dendritic surface area of the region of interest was measured to determine the number of clusters per 10 µm2. For each culture, we analyzed ~10 cells per experimental condition and ~100 clusters or ~15 spines per cell.
Metamorph software
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Sourced in France, United States
Metamorph software is a powerful image analysis and processing tool designed for use with a wide range of lab equipment. It provides a comprehensive suite of tools for image acquisition, enhancement, and measurement, enabling users to extract valuable data from their experiments and observations.
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Lab products found in correlation
Micromax, by Teledyne (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Dm6000, by Teledyne (2 mentions)
Coolsnap hq camera, by Teledyne (2 mentions)
Climabox, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Dm6000b, by Leica (1 mentions)
Axiovert 200m, by Teledyne (1 mentions)
Coolsnap camera, by Teledyne (1 mentions)
Lsm 710 meta, by Zeiss (1 mentions)
Imager m2 microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Axioscop microscope, by Zeiss (1 mentions)
Szx12, by Olympus (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Benchmark xt system, by Roche (1 mentions)
Thunder imaging system, by Leica (1 mentions)
Ultraview universal dab detection kit, by Roche (1 mentions)
25 protocols using metamorph software
1
Quantifying Neuronal Spine and Synaptic Protein Dynamics
2
Quantitative analysis of synaptic clusters
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Image acquisition was performed using a 63× objective (NA 1.32) on a Leica (Nussloch, Germany) DM6000 upright epifluorescence microscope with a 12-bit cooled CCD camera (Micromax, Roper Scientific) run by MetaMorph software (Roper Scientific, Evry, France). Image exposure time was determined on bright cells to obtain best fluorescence to noise ratio and to avoid pixel saturation. All images from a given culture were then acquired with the same exposure time and acquisition parameters. For cluster colocalization analysis, quantification was performed using MetaMorph software (Roper Scientific). For each image, several dendritic regions of interest were manually chosen and a user-defined intensity threshold was applied to select clusters and avoid their coalescence. For quantification of gephyrin or GABAAR synaptic clusters, gephyrin or receptor clusters comprising at least 3 pixels and colocalized on at least 1 pixel with VGAT clusters were considered. The number of clusters, the surface area and the integrated fluorescence intensities of clusters were measured. For surface expression analysis, quantification was performed using ImageJ (National Institutes of Health and LOCI, University of Wisconsin). Several dendritic regions of interest were manually chosen and mean average intensity per pixel was measured.
3
Quantifying Cell Migration in Wound Healing
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HeLa cells transfected with 2 μg pcD-empty, pcDCIRL-2 Wt or pcDCIRL-2 Mt. plasmids were seeded in 6-well plates at a density of 5 × 105 cells per well. When cells reached confluence, a scratch was performed with a sterile tip to create an artificial wound. Tiff format images were acquired every 6 h during 72 h using an inverted microscope (Leica DM 6000B). Cell migration from the wound edge into the wound space was recorded and a time-course quantification of the scratch width was evaluated using the Metamorph software (Roper Scientific). Cell wound repair was calculated using wound width (expressed in percentage of the initial size).
4
Live-cell and confocal microscopy of cells
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Following plating on gelatin-coated Lab-Tek I chambers, cells were maintained in phenol red-deprived DMEM-F12 (supplemented with low serum growth supplement and 10% FBS) for epifluorescence video microscopy experiments. For bright field video microscopy, cells were observed over a 30 min period using a Zeiss Axiovert 200M equipped with a Coolsnap camera (Roper Scientific, Trenton, NJ, USA). Cells were maintained at 37°C in 5% CO2 atmosphere using a temperature-controlled chamber (PECON, Erbach, Germany). Data were acquired using MetaMorph software (Roper Scientific). Timespan between image capture was 4 s. Confocal microscopy images of fixed cells were collected with a Zeiss (Oberkochen, Germany) LSM710 Meta confocal microscope using either a ×63 Plan Apochromat objective (oil immersion, 1.40 NA, DIC) at a 132 nm.pixel-1 resolution, leading to a slight XY oversampling. Optical slices were spaced by 1 μm so as to limit Z oversampling. Fluorescence filters were chosen and tested for each combination of plasmid construction and fluorescent dye so as to abolish spectral overlap. Zen software program was used to acquire images.
5
Immunofluorescence Staining Protocol
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Cells were plated on coverslips and transfected with the indicated plasmids. After 24–48 h, cells were washed with PBS, fixed with 3.7% formaldehyde in PBS for 20 min, then permeabilized with 0.1% Triton-X100 at room temperature (RT) for 4 min, or incubated with methanol at –20 °C for 10 min. Cells were incubated with 10 μg/mL of primary antibody (diluted in 0.2% gelatin in TBS) at RT for 2 h. After washes in TBS, coverslips were incubated with FITC- or TRITC- or Cy5-conjugated donkey-anti-mouse or donkey-anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories) at RT for 1 h. DNA was stained with Hoechst 33342 (1:10,000; Molecular Probes, Interchim) at RT for 10 min. After mounting with ProLong Gold Antifade reagent (Molecular Probes), images were acquired with a Zeiss Imager M2 microscope with the Apotome system and a Plan Apochromat 40×/1.3 DIC (oil) controlled by the ZEN software (Carl Zeiss Microscopy GmbH), or a Leica DMRA2 microscope (Rueil-Malmaison, France) equipped with an oil immersion ×100/1.4 apochromatic objective and a 12-bit Coolsnap FX CCD camera (Princeton Instruments, Roper Scientific, Evry, France) controlled by the MetaMorph software (Universal Imaging, Roper Scientific). Images were treated with ImageJ (https://imagej.nih.gov/ij/ ) or Adobe Photoshop (Adobe Sytems, Inc., San Jose, CA).
6
Quantification of Corticospinal Tract in Mouse Brains
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Fixed 129SvPas/C57Bl6-Thy1-eYFP brains were cut into 40-µm slices using a cryo-microtome (Microm, H560S); slices were mounted on Superfrost plus slides (Menzel-Gläser). Surface quantifications were performed on coronal slices. Epifluorescence of sections was digitized using an Axioscop microscope (Zeiss) equipped with a 5x dry objective and an EMCCD Quantum camera controlled by Metamorph software (Roper Scientific). ROIs were manually drawn and surface areas were calculated using ImageJ software. Since the cerebral volume for WT and KO mice was not the same21 (link), the area of the corticospinal tract was quantified on coronal brain sections selected based on morphometric landmarks. Thus, slice B corresponded to the end of the dentate gyrus (Bregma: −1.34 mm); slice C corresponded to the start of the habenular commissure (Bregma: −2.30mm); Slice D corresponded to the end of cerebral peduncle lateralization and the start of the rubrospinal tract (Bregma: −4.04 mm); and slice E corresponded to the 10th slice before the appearance of pyramidal tract decussation (Bregma: −7.56 mm). Epifluorescence for the pyramidal tract was further analyzed on sagittal sections using a confocal microscope (LSM 710, Zeiss), or in whole-mount brainstems using a stereo-microscope (Olympus SZX12).
7
High-Content Screening for Protein Localization
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We used the ArrayScan high‐content screening reader (XTI Live High
Content Platform, Thermo Fisher Scientific) for quantitative protein localization and
for the determination of fluorescence levels, or morphological changes in cells, in
accordance with published procedures (Fetz, Knauer, Bier, von Kries, & Stauber,
2009 ). One common step in our
analyses was the adjustment of the assay protocol, through the modification of
several parameters, to ensure optimal object identification, including background
correction, the setting of a threshold of pixel definition derived from the Hoechst
signal and object segmentation parameters. This step optimized object identification,
making it possible to exclude irregular “non‐cellular” objects automatically and to
quantify various cell parameters rapidly in thousands of cells. We then used the
following arrayscan protocols: “Spot detector” (mitochondrial marker, A20
aggresomes), “General measurement intensity” (IBA1, NLRP3 and A20 fluorescence
levels). Confocal microscopy was performed with a Leica Sp7 confocal microscope
(Leica, Wetzlar, Germany). MetaMorph software (Roper Scientific, Ottobrun, Germany)
was used for image acquisition
Content Platform, Thermo Fisher Scientific) for quantitative protein localization and
for the determination of fluorescence levels, or morphological changes in cells, in
accordance with published procedures (Fetz, Knauer, Bier, von Kries, & Stauber,
analyses was the adjustment of the assay protocol, through the modification of
several parameters, to ensure optimal object identification, including background
correction, the setting of a threshold of pixel definition derived from the Hoechst
signal and object segmentation parameters. This step optimized object identification,
making it possible to exclude irregular “non‐cellular” objects automatically and to
quantify various cell parameters rapidly in thousands of cells. We then used the
following arrayscan protocols: “Spot detector” (mitochondrial marker, A20
aggresomes), “General measurement intensity” (IBA1, NLRP3 and A20 fluorescence
levels). Confocal microscopy was performed with a Leica Sp7 confocal microscope
(Leica, Wetzlar, Germany). MetaMorph software (Roper Scientific, Ottobrun, Germany)
was used for image acquisition
8
Immunofluorescence analysis of brain slices
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Brain slices previously fixed with 4% PFA in PBS were incubated overnight at 4 °C with various primary antibodies diluted in incubation buffer (PBS containing 1% BSA and 3% Triton X-100) (Supplementary Table 1). Then, the slices were rinsed three times with PBS for 20 min and incubated with the same incubation buffer containing an adequate secondary antibody. Cell nuclei were visualized by incubating slices for 5 min with 1 µg/mL Hoechst 33258 in PBS. Fluorescent signals were observed with a Leica DMI 6000B microscope. The intensity profiles of immunolabeled slices were done using the Line scan tool of the Metamorph software (Roper Scientific, Evry, France). Control for nonspecific binding of the secondary antibody was performed by omitting the primary antibodies.
9
Agt Protein Localization in Placenta
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Immunohistochemical visualization of Agt-positive cells was carried out on six-micrometer sections obtained from paraffin-embedded material according to standardized protocols using an Agt-specific antibody (Supplementary Table S22 ). Before incubation, slices were submitted to deparaffinization which consisted of immersing the slides in successive baths of xylene (100%), ethanol (100–50%) and deionized water. Antigen retrieval was processed by microwave pretreatment in boiling 10 mM sodium citrate buffer (pH 6.0) for 10 min. Incubation of the primary antibody was performed for 60 min at room temperature using the Ventana Benchmark XT system. Slides were then processed with the Ultraview Universal DAB detection kit (Ventana). After 20×, 40× and 63× magnification acquisitions using a Leica Thunder Imaging System, high-resolution images were acquired in tiff format. Afterwards, Agt-positive intensity profiles were created based on villi thickness using the linescan tool of the Metamorph® software (Roper Scientific, Downingtown, PA, USA). After background correction, the area under the curve was calculated in the syncytiotrophoblast, giving access to a relative comparison with the intravillous space.
10
Quantitative Analysis of Synaptic Clusters
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Image acquisition was performed using a 63× objective (NA 1.32) on a Leica DM6000 upright epifluorescence microscope with a 12-bit cooled CCD camera (Micromax, Roper Scientific) run by MetaMorph software (Roper Scientific). Quantification was performed using MetaMorph software. Image exposure time was determined on bright cells to obtain the best fluorescence-to-noise ratio and avoid pixel saturation. All images from a given culture were then acquired with the same exposure time and acquisition parameters. For each image, several dendritic regions of interest were manually chosen, and a user-defined intensity threshold was applied to select clusters and avoid their coalescence. For quantification of gephyrin or GABAAR α2 synaptic clusters, gephyrin or receptor clusters comprising at least 3 pixels and colocalized on at least 1 pixel with VGAT clusters were considered. The integrated fluorescence intensities of clusters were measured.
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