Cells were first fixed (4% paraformaldehyde in phosphate-buffer saline (PBS), for 5 min at room temperature (RT)), then permeabilized (with 0.1% Triton X-100 in PBS, for 5 min, at RT), before blocked (with 10% BSA in PBS, for 2 h, at 4 °C). Cells were incubated with primary antibodies (in PBS with 5% BSA, overnight) and washed next in PBS. All secondary antibodies were first incubated for 1 h at RT in PBS, then washed, after that nuclei were counterstained with Hoechst. Slides were mounted on Mowiol mounting medium. Immunofluorescence microscopy was carried using an Olympus FV1000 (Olympus, Rungis, France) or a laser-scanning microscopy Nikon Ti2 coupled to a Yokogawa CSU-W1 head for confocal images.
Csu w1 head
Manufactured by Yokogawa
Sourced in France
The CSU-W1 head is a component of a laboratory equipment product line. It serves as a core functional unit, providing essential capabilities for the intended application. The description of the CSU-W1 head is limited to its core function, without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
4 protocols using csu w1 head
1
Immunofluorescence Microscopy Protocol
2
Live Imaging of Oxidative Stress Response
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For live imaging, animals were anesthetized in M9 containing 1mM levamisole and mounted between slide and coverslip on 3% agarose pads. Synchronized L4 animals were treated for 30min in a 96-well flat bottom plate, in 50μl of M9 containing 1mM or 10mM H2O2. Treated animals were transferred using a siliconized tip on a freshly seeded plate to recover, and imaged 1h30 to 2h later. Spinning-disk confocal imaging was performed on a system composed of an inverted DMI8 Leica microscope, a Yokogawa CSUW1 head, an Orca Flash 4.0 camera (2048*2018 pixels) piloted by the Metamorph software. Objective used were oil-immersion 40X (HC PL APO, NA 1.3) or 63X (HCX PL APO Lambda blue, NA 1.4). The temperature of the microscopy room was maintained at 20˚C for all experiments. Z-stacks of various body regions were acquired with a constant exposure time and a constant laser power in all experiments. Maximum intensity projections were used to generate the images shown. Fluorescence intensity measurements in int1, I2 and EPC cells were performed using the Fiji software, by manually drawing a region of interest (ROI) around the cell (int1, EPC), or applying a threshold (I2 neurons), background was subtracted and average pixel intensity was quantified.
3
Multi-Channel Fluorescence Microscopy
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Fluorescence images were acquired from cells in the exponential growth phase using a Zeiss Axio Observer 7 inverted microscope equipped with Zeiss Plan-Apochromat 63×/1.40 Oil DIC and Alpha Plan-Apochromat 100×/1.46 Oil DIC lenses coupled with a Spinning Disk Confocal Yokogawa CSU-W1 head. The system was equipped with solid-state excitation lasers (50 mW 515 nm [YFP] and 561 nm [RFPs]) and a DBP emission filter (527/54 + 645/60) from 3i (Intelligent Imaging Innovations). Device control and image acquisition were performed using SlideBook 6 software, and the cells were mounted in a Biopthec FCS2 chamber to enable temperature control during microscopy. When comparing fluorescent signals between two or more strains, all strains were placed next to each other but physically separated onto Glycine max lectin (L1395; Sigma-Aldrich) delimited drops on the coverslip. All images were processed using open-source Image J (Fiji) software (Schneider et al, 2012 (link)), and presented images correspond to maximal or sum projections as indicated in each figure legend.
4
Live Imaging of Hydrogen Peroxide Effects
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For live imaging, animals were anesthetized in M9 containing 1mM levamisole and mounted between slide and coverslip on 3% agarose pads. Synchronized L4 animals were treated for 30min in a 96-well flat bottom plate, in 50µl of M9 containing 1mM or 10mM H2O2. Treated animals were transferred using a siliconized tip on a freshly seeded plate to recover, and imaged 1h30 to 2h later. Spinning-disk confocal imaging was performed on a system composed of an inverted DMI8 Leica microscope, a Yokogawa CSUW1 head, an Orca Flash 4.0 camera (2048*2018 pixels) piloted by the Metamorph software. Objective used were oilimmersion 40X (HC PL APO, NA 1.3) or 63X (HCX PL APO Lambda blue, NA 1.4). The temperature of the microscopy room was maintained at 20 ˚C for all experiments. Z-stacks of various body regions were acquired with a constant exposure time and a constant laser power in all experiments. Maximum intensity projections were used to generate the images shown. Fluorescence intensity measurements in int1, I2 and EPC cells were performed using the Fiji software, by manually drawing a region of interest (ROI) around the cell (int1, EPC), or applying a threshold (I2 neurons), background was subtracted and average pixel intensity was quantified.
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