Brains were post-fixed in 4% PFA overnight in 4°C, protected from light and then washed in PBS for 24 h. Brains were dissected and the ipsilateral injured cortex was placed in a 30 mL glass amber packer bottle (VWR; Suwanee, GA) for the remaining steps. A four-step dehydration procedure was performed with increasing concentrations of tetrahydrofuran (THF) in distilled water: (1) 50% THF for 2 h; (2) 80% THF for 2 h; (3) 100% THF overnight (~16 hours); (4) 100% THF for 24h. All incubation steps were done at 4°C on a shaker. For clearing, the brains were incubated in a 1/3 benzyl alcohol (Sigma-Aldrich, 402834), 2/3 benzyl benzoate (Sigma-Aldrich, B6630) (BABB) solution for 2–4 h (on a shaker at 4°C) prior to imaging. Cleared cortices were imaged on a LaVision BioTec Ultramicroscope based around an Olympus MVX10 zoom microscope body (variable zoom 0.63 – 6.3 X) including light sheet excitation lines OPSL (50mW), 488nm, OPSL (50mW), 561nm and diode laser (50mW), 647nm. The camera used was an Andor Neo scientific low noise sCMOS camera with GFP/FITC: 525/50, TRITC/Alexa594: 620/60 and Alexa 647/Cy5: 700/80 detection channels. 3D image analysis was done using Imaris 8.1.2 (Bitplane) software.
Mvx10 zoom microscope body
Manufactured by Olympus
Sourced in Japan, Germany
The MVX10 zoom microscope body is a versatile optical instrument designed for a range of applications. It features a zoom function that allows for continuous magnification adjustment, enabling users to observe specimens at various levels of detail. The MVX10 provides high-quality imaging capabilities and is suitable for use in various research and analysis settings.
Automatically generated - may contain errors
Lab products found in correlation
Lavision biotec ultramicroscope 2, by Miltenyi Biotec (2 mentions)
Ultramicroscope, by Miltenyi Biotec (2 mentions)
Lsm 710, by Zeiss (2 mentions)
B6630, by Merck Group (1 mentions)
Neo scmos camera, by Oxford Instruments (1 mentions)
Fluar 5 0.25 objective, by Zeiss (1 mentions)
Fluar 10 0.5 objective, by Zeiss (1 mentions)
7 protocols using mvx10 zoom microscope body
1
High-Resolution 3D Imaging of Cleared Brain
2
Large-Scale 3D Imaging of Cleared Samples
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Optically cleared samples were imaged on a LaVision BioTec Ultramicroscope II (LaVision BioTec, Bielefeld, Germany) with an Olympus MVX10 Zoom Microscope Body (Olympus, Tokyo, Japan) with an optical magnification range from 1.26x to 12.6x and an NA of 0.5. An NKT SuperK (Power SK PP485) supercontinuum white light laser served as excitation light source. For excitation and emission detection of specific fluorophores custom band-pass filters (excitation 470/40, 577/25 or 640/30 nm; emission 525/50, 632/60 or 690/50 nm) in combination with an Andor Neo sCMOS Camera with a pixel size of 6.5 x 6.5 μm2 were used. For image acquisition, Z-steps 3 μm were chosen and tile scanning was performed with an overlap of 20%.
3
Whole-Organ Light Sheet Microscopy
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Large samples (such as brain, spinal cord, CNS, kidney, and muscle) were imaged using a light sheet fluorescence microscope (Ultramicroscope, LaVision BioTec, Germany) equipped with an sCMOS camera (Andor Neo), a 2×/0.5 objective lens equipped with a dipping cap, and an Olympus MVX10 zoom microscope body (magnification range of ×0.63 to ×6.3). The cleared tissues were mounted on the sample holder and incubated with the final clearing solution (such as DBE) in the sample reservoir. For entire scanning of whole organs, the z-step interval was 5 or 10 μm, and for image acquisition in the regions of interest, an interval in the range of 2 to 5 μm was used.
4
Ultramicroscopy Imaging of Optically Cleared Samples
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Optically cleared samples were imaged on a LaVision BioTec Ultramicroscope II (LaVision BioTec, Bielefeld, Germany) equipped with an Olympus MVX10 Zoom Microscope Body (Olympus, Tokyo, Japan) allowing an optical magnification range from 1.26x to 12.6x and an NA of 0.5. An NKT SuperK (Power SK PP485) supercontinuum white light laser served as excitation light source. For excitation and emission detection of specific fluorophores custom band-pass filters (excitation 470/40, 577/25 or 640/30 nm; emission 525/50, 632/60 or 690/50 nm) in combination with an Andor Neo sCMOS Camera. For image acquisition, Z-steps 3 μm were chosen. 3D reconstruction and analysis of ultramicroscopy stacks were performed by using the volume rendering software Voreen [39 (link), 56 (link)].
5
LSFM Imaging of Cleared Tissue Samples
6
Spatial Mapping of Motor Neuron Projections in Skeletal Muscles
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To obtain the spatial distribution of the MEPs in skeletal muscles, the cleared muscles were imaged using a light sheet fluorescence microscope (LaVision BioTec I, Bielefeld, Germany) equipped with an sCMOS camera (Andor Neo), a ×2/0.5 objective lens equipped with a dipping cap, and an Olympus MVX10 zoom microscope body (magnification range of ×0.63–×6.3). The cleared tissues were mounted on the sample holder and incubated with DBE in the sample reservoir. The z-step interval was 5 μm.
To analyze the labeled motor neurons or the intramuscular diffusion of the CTB, the whole spinal cord or muscle slices were imaged with inverted confocal fluorescence microscopy (LSM710; Zeiss, Oberkochen, Germany) equipped with Fluar ×5/0.25 objective (dry, W.D. 12.5 mm) and Fluar ×10/0.5 objective (dry, W.D. 2.0 mm). The cleared spinal cord segments or muscle slices were placed on a coverslip with the clearing reagent (DBE) or 0.01 M PBS, with another coverslip being put on the top of the sample.
To analyze the labeled motor neurons or the intramuscular diffusion of the CTB, the whole spinal cord or muscle slices were imaged with inverted confocal fluorescence microscopy (LSM710; Zeiss, Oberkochen, Germany) equipped with Fluar ×5/0.25 objective (dry, W.D. 12.5 mm) and Fluar ×10/0.5 objective (dry, W.D. 2.0 mm). The cleared spinal cord segments or muscle slices were placed on a coverslip with the clearing reagent (DBE) or 0.01 M PBS, with another coverslip being put on the top of the sample.
7
Imaging Neuromuscular Junctions with LSFM
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Transparent muscles were imaged with the light-sheet fluorescence microscope (LSFM, UltraMicroscope, LaVision BioTec, Germany) equipped with an sCMOS camera (Andor Neo), a 2×/0.5 objective lens furnished with a dipping cap, and an Olympus MVX10 zoom microscope body (magnification range of ×0.63 to ×6.3). For NMJs labeled with α-BTX 647 and nerve branches with YFP, 633 nm and 488 nm were applied, respectively, as the exciting wavelengths. The z step size was set to 3 μm for neonatal muscles and 5 μm for adult muscles. Images of the samples were acquired for subsequent processing and analysis after the appropriate setting of the imaging parameters.
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