After fiber photometry recordings, pharmacological experiments, ChR2 or GtACR2 optogenetics experiments, animals were euthanized with pentobarbital (300 mg/kg) and perfused with ringer’s solution followed by 4% PFA (antigenfix, F/P0014, MM France) at 4 °C. The brains were extracted and left in 4% PFA overnight at 4 °C, for post fixation. Then, they were transferred to a 30% sucrose solution, in PBS 1× at 4 °C, for cryoprotection. After sinking in the sucrose solution, the brains were embedded in optimum cutting temperature compound (OCT) and sliced into 100-50 µm coronal slices, in a freezing sliding microtome (12062999, Thermo Scientific). Sections were incubated with a DNA-specific fluorescent probe Hoechst33342 (1:5000, Fischer scientific) for 20 min, washed with PBS 1× followed by mounting on microscope slides with Polyvinyl alcohol PVA-DABCO mounting medium (Sigma Aldrich, France). Images were acquired at the fluorescence microscope (THUNDER Imager 3D Tissue, Leica), using the de-convolution system.
Thunder imager 3d tissue
Manufactured by Leica
The THUNDER Imager 3D Tissue is a high-performance microscope system designed for three-dimensional (3D) imaging of tissue samples. It utilizes advanced optical technology to capture clear, high-resolution images of complex biological structures within thick tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
K5 scmos camera, by Leica (1 mentions)
Metamorph 7, by Molecular Devices (1 mentions)
Dmi6000b inverted microscope, by Leica (1 mentions)
Tetramisole, by Merck Group (1 mentions)
Axiovert, by Yokogawa (1 mentions)
Application suite x software, by Leica (1 mentions)
4 protocols using thunder imager 3d tissue
1
Brain Tissue Preparation for Imaging
2
Imaging Ciliated Neurons in C. elegans
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L1 and L3 hermaphrodite larvae and one-day-old adults were immobilized in 10mM tetramisole hydrochloride (MP Biomedicals) and mounted on 10% agarose pads placed on microscope slides. Animals were imaged on an upright THUNDER Imager 3D Tissue (Leica). Complete z-stacks of ciliated neurons (ASH, AWA, AWB, and AWC) were acquired at 0.22μm intervals with K5 sCMOS camera (Leica) in Leica Application Suite X software using an HC Plan Apochromat 63X NA 1.4–0.60 oil immersion objective. For BiFC and experiments examining ODR-3::TagRFP levels in wild-type and ric-8(md1909) mutant AWC neurons (Figure 5 ), adult animals were imaged on an inverted Nikon Ti-E microscope with Yokogawa CSU-X1 spinning disk confocal head using 60X NA 1.40 oil immersion objective. Complete z-stacks of either AWC cilia or entire AWC neurons were acquired in MetaMorph 7 (Molecular Devices).
3
Immunofluorescence Imaging of Cellular Structures
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Cells were fixed with formaldehyde (4%, 10 min), permeabilized with Triton X-100 (0.05%, 5 min), and blocked for 1 h with a mixture of normal donkey serum (2%) and fetal bovine serum (10%) diluted in DPBS. Cells were incubated overnight with the adequate primary antibody at 4°C followed by incubation for 2 h at room temperature with the adequate secondary antibody. Cells were counterstained with DAPI (1 µg/ml, 10 min) to label nuclei, and imaged using the Leica DMI6000 B inverted microscope, the Leica SP8 MP confocal multiphoton microscope or the Leica THUNDER Imager 3D Tissue. In PXN-labeled cells (Fig. 7 E and Fig. S5 A ), the number of focal adhesion complexes was evaluated after image background subtraction using the Find Maxima function of the ImageJ software. Radial intensity profile of the β1 integrin signal (Fig. 5 ) was produced using the Clock-scan plug-in (Dobretsov and Romanovsky, 2006 (link)). The number of TGN46-positive vesicles was obtained using the ComDet plug-in of the ImageJ software.
4
Quantifying Neuronal Morphology in C. elegans
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Larvae or one-day old adult hermaphrodites were immobilized in 10 mM tetramisole (Sigma) and mounted on 10% agarose pads set on microscope slides. Animals were imaged on an inverted spinning disk confocal microscope (Zeiss Axiovert with a Yokogawa CSU22 spinning disk confocal head). Complete z-stacks of A/PQR neurons were acquired at 0.27 µm intervals in Slidebook 6.0 (3i -Intelligent Imaging Innovations) using a Plan Apochromat 100X NA 1.4 or 63X NA 1.4 oil immersion objective. For experiments examining cilia position and GRDN-1 localization in the lin-44 mutant background, adult or larval animals were imaged on an upright THUNDER Imager 3D Tissue (Leica). Complete z-stacks of PQR were acquired at 0.22 µm intervals in Leica Application Suite X software using an HC Plan Apochromat 63X NA 1.40-0.60 oil immersion objective. Acquired optical sections were rendered into maximum intensity projections using Slidebook 6.0 or Image J (NIH, Bethesda, MD). A/PQR morphology and localization of fluorophore-tagged proteins were quantified in Slidebook 6.0 or Image J.
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