For observation of neuronal morphology, images were captured under a confocal microscopy (excitation wavelength, 488 and 543 nm) and multiple planes were combined to Z-projections (Nikon, Tokyo, Japan). For axon tracing, serial sections were mounted in sequence and images were automatically captured under a stereomicroscope (Keyence, Osaka, Japan) using a tiling protocol. Axonal fibers were reconstructed in two dimensions section by section; serial images were superimposed, and neuronal processes were traced using ImageJ software (National Institutes of Health, Bethesda, MD, United States). For quantitative analysis of VAMP2-mOrange localization, VAMP2-mOrange positive puncta were defined by combining the threshold (between the intensity values of 60 to 255) and the length (above 2 μm) to distinguish from the background signal. The number of VAMP2-mOrange puncta was counted per dendritic or axonal segment and compared between dendrites and axons (Mann–Whitney U test). All statistical values obtained from morphological experiments are presented as means ± standard error of the mean (SEM).
Stereomicroscope
Manufactured by Keyence
Sourced in Japan
The Stereomicroscope is a binocular optical microscope that provides a three-dimensional view of the specimen. It is designed to observe and inspect samples at low magnifications, typically ranging from 10x to 40x. The stereomicroscope uses two separate optical paths, one for each eye, to create a stereoscopic image that gives the observer a sense of depth and spatial awareness of the subject matter.
Automatically generated - may contain errors
3 protocols using stereomicroscope
1
Quantifying Neuronal Morphology and VAMP2 Localization
2
In Situ Hybridization of Zebrafish Embryos
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In situ hybridization (ISH) probes for MSANTD2 were cloned from WT zebrafish cDNA by PCR using the GoTaq polymerase (Promega). PAX2A and HER5 probes were given by Dr. Sebastian Dworkin lab, La Trobe University, Melbourne, Australia.
Zebrafish embryos were collected, removed from their chorion, sorted and fixed in paraformaldehyde (PFA) 4%, dehydrated in methanol and stored at—20°C. ISH was performed following the Thisse Lab protocol (Thisse and Thisse 2008 (link); 2014 (link)). Embryos were rehydrated and washed in phosphate buffered saline (PBS)—Tween (PBT) solution. They were permeabilized with proteinase K and fixed in PFA. Each embryo was incubated with probes overnight at 65°C in hybridization mix supplemented with 5% Dextran Sulfate (Millipore). Nonhybridized probes were removed with several washes in formamide and saline-sodium-citrate solutions. Embryos were incubated overnight at 4°C with α-DIG (digoxigenin) antibodies (Roche). Nonfixed antibodies were removed with PBT washes. Probes were revealed with nitro blue tetrazolium chloride - 5-bromo-4-chloro-3-indolyl-phosphate (NBT-BCIP) (Roche). Embryos were fixed in PFA. After removing of the background with ethanol bath, embryos were stored in glycerol 80% at 4°C. Pictures were taken under Leica stereomicroscope and Keyence VHX-7000 microscope.
Zebrafish embryos were collected, removed from their chorion, sorted and fixed in paraformaldehyde (PFA) 4%, dehydrated in methanol and stored at—20°C. ISH was performed following the Thisse Lab protocol (Thisse and Thisse 2008 (link); 2014 (link)). Embryos were rehydrated and washed in phosphate buffered saline (PBS)—Tween (PBT) solution. They were permeabilized with proteinase K and fixed in PFA. Each embryo was incubated with probes overnight at 65°C in hybridization mix supplemented with 5% Dextran Sulfate (Millipore). Nonhybridized probes were removed with several washes in formamide and saline-sodium-citrate solutions. Embryos were incubated overnight at 4°C with α-DIG (digoxigenin) antibodies (Roche). Nonfixed antibodies were removed with PBT washes. Probes were revealed with nitro blue tetrazolium chloride - 5-bromo-4-chloro-3-indolyl-phosphate (NBT-BCIP) (Roche). Embryos were fixed in PFA. After removing of the background with ethanol bath, embryos were stored in glycerol 80% at 4°C. Pictures were taken under Leica stereomicroscope and Keyence VHX-7000 microscope.
3
Photographing and Dissecting Ethanol-Preserved Shells
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Ten to 30 photographs were taken of each shell using Keyence LHX5000 digital microscope and merged to create a single image using Photoshop. The ethanol-preserved specimen was dissected under a Zeiss stereomicroscope, and photographs were taken using a Keyence LHX5000 digital microscope. Abbreviations: HA: Collection András Hunyadi, (Budapest, Hungary); HE: Collection Christa Hemmen (Wiesbaden, Germany); HNHM: Hungarian Natural History Museum (Budapest, Hungary); MNHN: Muséum National d'Histoire Naturelle (Paris, France).
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