For single-cell gliding on glass, cells were grown in CYE at 28°C to an A600 nm ≈ 0.7. Cells were diluted to an A600 nm ≈ 0.05 and 100 μL were spotted into μ-Slide chambers with glass coverslip bottom (Ibidi). After 5-minute incubation, floating cells were washed out with fresh CYE medium and gliding of adherent cells was monitored by phase contrast microscopy on a Nikon Eclipse TE-2000 microscope equipped with a 100× NA 1.3 Ph3 objective, a perfect focus system to maintain the plane in focus, and an Orcaflash 4.0 LT digital camera (Hamamatsu Photonics, Shizuoka, Japan). GldL-alfa/NBalfa-sfGFP localization was observed by Hilo microscopy. Cells were grown in CYE overnight without shaking at 28°C. NBalfa-sfGFP expression was induced with 1 mM IPTG for 1 hour prior to observation. Cells were spotted on a 2% low-melting agarose pad for immediate observation. Hilo fluorescence microscopy and FRAP experiments were performed with a Nikon Eclipse Ti2 microscope equipped with a 100x NA 1.45 Ph3 objective, an Orca-Fusion digital camera (Hamamatsu Photonics), a perfect focus system, and an Ilas2 TIRF/FRAP module (Gataca Systems, Massy, France).
Orca flash 4.0 lt digital camera
Manufactured by Hamamatsu Photonics
Sourced in Japan
The ORCA-Flash 4.0 LT digital camera is a scientific imaging device designed for a variety of applications. It features a CMOS image sensor with a resolution of 4.2 megapixels and a pixel size of 6.5 μm. The camera is capable of capturing images at a maximum frame rate of 100 frames per second. It supports various bit depths and readout modes to accommodate different imaging requirements.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Bx63 stereomicroscope, by Olympus (2 mentions)
Cellsens dimension 1, by Olympus (2 mentions)
Glass coverslip bottom, by Ibidi (1 mentions)
Nikoneclipse ti2 microscope, by Hamamatsu Photonics (1 mentions)
μ slide chamber, by Ibidi (1 mentions)
Eclipse te2000 microscope, by Nikon (1 mentions)
Orca fusion digital camera, by Hamamatsu Photonics (1 mentions)
Dmire2 microscope, by Leica (1 mentions)
Cm1860, by Leica (1 mentions)
Imager z2 microscope, by Hamamatsu Photonics (1 mentions)
Cm1860 cryostat, by Leica (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
Lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Pdgfrα, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 fluorescence microscope, by Zeiss (1 mentions)
Duolink in situ orange starter kit mouse rabbit, by Merck Group (1 mentions)
Zen software, by Zeiss (1 mentions)
Prism 6, by GraphPad (1 mentions)
10 protocols using orca flash 4.0 lt digital camera
1
Single-cell Gliding Motility Assay
2
Fluorescence Microscopy for Cellular Imaging
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Brightfield or fluorescent images were obtained on a Leica DMIRE2 microscope through an ORCA-flash 4.0 LT+ digital camera (model C11440, Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany). Hoechst 33,258 and PI were visualized through A4 and N21 filter cubes, respectively. ImageJ software was utilized to process the captured images.
3
Phospho-mTOR Expression in Fetal Brain Sections
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Fetal brains were sectioned at 8 μm in a coronal plane using a Leica CM1860 cryostat (Leica Biosystems, Buffalo Grove, IL). Sections were fixed with ice-cold methanol (30 min, −20°C), rinsed in PBS, incubated in 10% normal serum (60 min), followed by incubation with Phospho-mTOR (Ser2448) (1:250; #2971) primary antibody overnight at 4°C in a humidified chamber. The sections were further incubated with goat anti-rabbit IgG secondary antibody (Alexa Fluor 488, #A11008; Invitrogen, Carlsbad, CA), for 60 min at room temperature. Nuclei were stained with DAPI (ProLong Gold antifade, #P36931; Invitrogen, Carlsbad, CA). Digital images were captured using an Olympus BX63 stereomicroscope equipped with U-HGLGPS fluorescent light source, ORCA-Flash 4.0 LT digital camera (Hamamatsu, Japan), and Olympus cellSens Dimension 1.16 software (Olympus, Japan).
4
High-throughput Fluorescence Microscopy Imaging
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Imaging was performed with a Zeiss Imager Z2 microscope equipped with a Hamamatsu ORCA-flash4.0 LT digital camera using a 20× objective and manual focusing in live view. Three none overlapping images were acquired from each well and used to derive the data. Exposure times were in the range of 0.5–2 s for the different fluorescence channels. Imaging conditions were kept constant for each fluorescence channel in each experiment, and carefully adjusted not to include overexposed areas. No adjustments were made to the images before image analysis and data retrieval using the CellProfiler software (15 (link)). Brightness and contrast were adjusted for visualization purposes in the figures but kept constant within each experiment.
5
Immunostaining Fetal Brain Sections
6
Imaging and Analyzing Hippocampal Neurons
7
CXCR7 Immunofluorescence Staining
8
Proximity Ligation Assay for Protein Interactions
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PLA was performed with the Duolink in situ orange starter kit mouse/rabbit (Sigma-Aldrich Corp.) according to the manufacturer's instructions for custom solutions. Briefly, E13.5 embryos were fixed in 4% PFA in PBS and infiltrated with 30% sucrose in PBS before being mounted in OCT compound (Sakura Finetek USA, Inc.). Sections (8 µm) deposited on glass slides were fixed in 4% PFA in PBS with 0.1% Triton X-100 for 10 min and washed in PBS with 0.1% Triton X-100. Sections were blocked for 1 h in 5% normal donkey serum in PBS and incubated overnight at 4°C in primary antibodies diluted in 1% normal donkey serum in PBS. The following antibodies were used for PLA: PDGFRα (1:100; Thermo Fisher Scientific) and PDGFRβ (1:25; 28; Becton Dickinson). Sections were washed in PBS prior to incubation in PLA probes diluted in 1% normal donkey serum in PBS. Sections were photographed using an ORCA-Flash4.0 LT digital camera (Hamamatsu Photonics K.K.) fitted onto an AxioObserver.Z1 fluorescence microscope (Carl Zeiss, Inc.). Extended depth of focus was applied to z-stacks using Zen software (Carl Zeiss, Inc.) to generate images with the maximum depth of field. The number of puncta per 10 (mesenchyme) and five (epithelium) 50-µm2 areas were counted in two independent experiments, and statistical analyses were performed with Prism 6 (GraphPad Software, Inc.) using a two-tailed unpaired t-test.
9
Immunohistochemical Analysis of DUSP1, p38, and p-p38 in Human Cardiac Tissue
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For assessment of DUSP1, total p38, and phosphorylated p38 expression, FFPE sections of human cardiac tissue were used. Paraffin was removed in xylene and the sections were dehydrated in a graded ethanol series. Primary antibodies were diluted in blocking buffer and incubated on tissue sections overnight at 4°C. Primary antibodies used were as follows: 1:50 DUSP1 (sc-373841, Santa Cruz), 1:50 p-p38 MAPK (Thr180/Tyr182) (#9211, Cell Signaling Technology), and 1:100 p38 MAPK (#9212, Cell Signaling Technology). The sections were viewed with a Nikon Eclipse 80i microscope (Nikon, Melville, NY, USA), and captured with an ORCA-Flash4.0 LT digital camera (Hamamatsu, Hamamatsu City, Japan) and NIS-Elements AR v4.30.01 software (Nikon, Melville, NY, USA).
10
Quantification of Tubulin Modifications in Spastin-Expressing Cells
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Cells were fixed with 4% paraformaldehyde-sucrose for 20 min at room temperature and then processed for immunofluorescence. Average intensity of acetylated, tyrosinated or β-tubulin in single HeLa cells was measured by using Metamorph software (Roper Scientific, Evry, France) in pre-selected cells expressing mild levels of GFP-tagged spastin and having good morphology. Data were normalized to the average intensity of tubulins of untransfected and/or untreated cells. Each experiment was repeated between two and six times. The number of cells analyzed ranged between 40 and 200. For colocalization analysis of GFP-tagged spastin with calreticulin or M1WT-Flag, images were acquired with a Leica DM4B microscope equipped with light fiber source, a 63×/1.3 NA HC PL Fluotar oil-immersion objective, a 1.6× tube lens and an Orca Flash 4.0 LT digital camera (Hamamatsu, Massy, France). Each channel was then processed for background subtraction and 2D deconvolution with Metamorph software in order to improve the signal-to-background ratio. Images were then analyzed with the threshold colocalization plugin in ImageJ (http://imagej.nih.gov/ij/ ). Data are the mean from 10-35 cells. Statistical significance was determined using GraphPad Prism software.
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