SYTOX nuclear green stain is impermeable to living cells, but stains nuclei in a syncitium (or otherwise following membrane degradation) (Goonesinghe et al., 2012 (link)). To visualise migration of YSL nuclei relative to the blastoderm margin during epiboly, embryos from heterozygous parents were injected with 1 nl of 0.5 mM Sytox Green fluorescent nucleic acid dye (Invitrogen, United States) into the yolk cell at 3 hpf and then visualised at 4 hpf and 6 hpf under a fluorescence stereomicroscope (Zeiss). Embryos were kept in E3 medium for genotyping verification. Images were captured and processed using a Zeiss AxioCam MR and AxioVision 4.5 software.
Fluorescence stereomicroscope
Manufactured by Zeiss
Sourced in United States, Germany
The Fluorescence Stereomicroscope is an optical instrument designed for the observation and analysis of fluorescently labeled samples. It combines the capabilities of a stereomicroscope with the ability to detect and visualize fluorescent signals, enabling the study of biological specimens, materials, and other samples that exhibit fluorescence properties.
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Lab products found in correlation
Axiovision 4, by Zeiss (1 mentions)
Axiocam mr, by Zeiss (1 mentions)
Pcdh ef1 mcs t2a copgfp vector, by System Biosciences (1 mentions)
Lightsheet z 1 microscope, by Zeiss (1 mentions)
Axio zoom v16, by Zeiss (1 mentions)
Axiocam 506 color camera, by Zeiss (1 mentions)
Arivis software, by Zeiss (1 mentions)
5 protocols using fluorescence stereomicroscope
1
Visualizing Yolk Syncytial Layer Nuclei Migration
2
Establishment and Isolation of Metastatic E0771GFP Cell Lines
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To establish the E0771GFP cell line, E0771 cells were stably transduced with a GFP-expressing lentiviral vector (pCDH-EF1-MCS-T2A-copGFP vector; System Biosciences) as described previously (48 (link)). GFP+ cells were then FACS-sorted, expanded in tissue culture, and inoculated (106 cells) into the fourth mammary fat pad of a C57BL/6J female. After 28 days, the mouse was euthanized, and two distinct GFP+ lung metastatic foci were dissected under a fluorescence stereomicroscope (Zeiss). Recovered tissue was then dissociated and digested with collagenase I at 37°C for 45 min. The cell suspensions were then filtered through a 70-μm cell strainer and plated in 10-cm dishes containing RPMI 1640 supplemented with 10% calf serum with iron and 1% penicillin-streptomycin. Tumor cells were then expanded in tissue culture, FACS-sorted on the basis of GFP expression, and maintained as the M11GFP and M12GFP cell lines.
3
In Vivo Zebrafish Tumor Imaging
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Live embryos were photographed at 1 h (t0, 48 hpf), 1 day (t1) and 4 days (t4) post implantation on agarose-coated dishes using an AxioZoom V16 fluorescence stereomicroscope (Zeiss, Oberkochen, Germany, EU) equipped with a digital Axiocam 506 color camera (Zeiss). The mean area of the tumor was manually measured using FIJI software [24 (link)]. Light sheet microscopy experiments were performed using a Light Sheet Z.1 microscope (Zeiss). For this purpose, t0, t1, and t4 embryos were embedded in a low melting agarose cylinder (1% low melting agarose:fish water, 1:1) and immersed in the observation chamber filled with fish water and anesthetic. Maximum intensity projections were obtained using the Zen software (Zeiss) and 3D reconstructions were made after z-stack processing with Arivis software (Zeiss).
To detect apoptotic cells, 48 hpf embryos were microinjected in the eye with 2.0 nL of a solution containing the anticancer drug under testing. After injection, zebrafish embryos were grown at 33 °C for 4 days. At t4, live embryos were soaked in fish water containing 2 μg/mL acridine orange and incubated at 28 °C for 20 min. After 8 washes for 5 min each with fish water, embryos were anesthetized and analyzed immediately with a fluorescence stereomicroscope (Zeiss).
To detect apoptotic cells, 48 hpf embryos were microinjected in the eye with 2.0 nL of a solution containing the anticancer drug under testing. After injection, zebrafish embryos were grown at 33 °C for 4 days. At t4, live embryos were soaked in fish water containing 2 μg/mL acridine orange and incubated at 28 °C for 20 min. After 8 washes for 5 min each with fish water, embryos were anesthetized and analyzed immediately with a fluorescence stereomicroscope (Zeiss).
4
Zebrafish Maintenance and Fluorescent Embryo Selection
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Zebrafish were maintained under 14 h light/10 h dark cycles at 28°C in groups of 10–30 adults according to legal regulations (EU-Directive 201_63). We used standard procedures to cross zebrafish for egg production and to prepare them for experimental analysis (Weber, et al., 2016 (link)). Using a fluorescence stereomicroscope (Zeiss, Germany), embryos expressing the expected fluorescence patterns were selected and kept in separate dishes. Usually we maintained up to 20 embryos from one to three different clutches for an experimental session. In all sessions we used sibling controls.
5
Mapping Neuronal Inactivation in Mice
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All the mice in the inactivation experiment received, after completion of the behavioral procedure, intra-dBNST bilateral infusions (100 nl per side) of muscimol conjugated with a fluorophore (BODIPY-TMR-X muscimol conjugate, M23400, Invitrogen, 5 mM in aCSF). The mice were rapidly decapitated 45 min later, and their brains were immediately placed in 4% paraformaldehyde (PFA) in 1 × PBS (pH 7.4) for fixation at 4°C overnight. The brains were then stored in 1 × PBS and cut on a vibratome into 60-μm coronal sections. The sections were mounted on slides and observed at Zeiss fluorescence stereomicroscope for verifying the location of the Muscimol-BODIPY intracerebral infusion.
The mice in the optogenetic and calcium experiments were killed after completing the behavioral procedure, and their brains were processed for histological analysis as described above. After vibratome sectioning, the sections were counterstained with DAPI and mounted on slides. Images were acquired using a confocal microscope and a Mirax slide scanner.
The mice in the optogenetic and calcium experiments were killed after completing the behavioral procedure, and their brains were processed for histological analysis as described above. After vibratome sectioning, the sections were counterstained with DAPI and mounted on slides. Images were acquired using a confocal microscope and a Mirax slide scanner.
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