Embryonic or postnatal mouse brains were fixed, respectively, by immersion in or intracardial perfusion with 4% PFA. Labeling of callosal projection neurons was achieved by placing DiI crystals (Molecular Probes, Life Technologies) in the cingulate or somatosensory cortex. The brains were returned to 4% PFA, placed in light-tight containers, and incubated at 37°C or at room temperature for 3–5 weeks. The brains were then embedded in 3% agarose and sectioned using a vibratome (Leica Microsystems).
Dii crystal
Manufactured by Thermo Fisher Scientific
Sourced in United States
DiI crystals are a fluorescent lipophilic dye used for labeling and tracking cell membranes. They are widely used in cellular and molecular biology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Evos fl inverted microscope, by Thermo Fisher Scientific (2 mentions)
Alexafluor 647 conjugate ctb, by Thermo Fisher Scientific (2 mentions)
Cytotune emgfp sendai fluorescence reporter, by Thermo Fisher Scientific (2 mentions)
D3911, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Vibratome, by Leica (1 mentions)
Tcs sp, by Leica (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Bromodeoxyuridine (brdu), by Bio-Rad (1 mentions)
Freezing microtome, by Leica (1 mentions)
Vibratome vt1000, by Leica (1 mentions)
Vt1200, by Leica (1 mentions)
Dissecting microscope, by AmScope (1 mentions)
Tcs sp2 confocal laser scanning microscope, by Leica (1 mentions)
Paraformaldehyde solution, by Electron Microscopy Sciences (1 mentions)
Vectashield hardset mounting medium, by Vector Laboratories (1 mentions)
Vt1200 vibratome, by Leica (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Smi312, by BioLegend (1 mentions)
Triton x 100, by Merck Group (1 mentions)
18 protocols using dii crystal
1
Tracing Callosal Projection Neurons
2
Neuronal Migration Tracing with DiI
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To investigate neuronal migration, we used a glass micropipette to place DiI crystals (Molecular Probes; Lavdas et al., 1999 (link); E14, n = 3; E16, n = 5) on one hemisphere of cultured slices. After tracer placement, slices were cultured in Neurobasal medium for a further 48 h and were then fixed for 3 h in 4% formaldehyde in PBS. Slices were subsequently rinsed in PBS, mounted on microscope slides, and observed with a laser-scanning confocal microscope (Leica TCS SP).
3
Multimodal Neurotracing in Embryos
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For DCN neurotracing, embryos were dissected, the spinal cord was exposed via dorsal laminectomy, fixed in 4% paraformaldehyde (PFA) for 2 h, and washed in PBS. For spinal DCN neurotracing, DiI crystals (Molecular Probes) were placed in the dorsolateral region of the spinal cord using glass micropipettes (Renier et al., 2010 (link)). For supraspinal neurotracing, the skull was removed, and DiI crystals were placed into the caudal hindbrain. For sensory DCN neurotracing, DiI crystals were placed in dorsal root ganglia and the ventral roots were cut. Tissue samples were placed in 4% PFA at 37°C for up to 5 weeks for DiI diffusion. Samples were then washed with PBS and embedded in 4–6% agarose for vibratome sectioning (200 μm). Sections were mounted with PBS for confocal microscopy.
4
Thalamocortical Axon Tracing Using DiI
5
Developmental Tracing of Neural Circuits
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For BrdU labeling, time-mated females received one intraperitoneal (i.p.) injection with BrdU (100 mg/kg; Sigma, St.Louis, Missouri, USA,) at either 12.5 days post coitum (d.p.c.) or E14.5. Embryos were collected at the indicated times after BrdU administration in 4% PFA. After cryoprotection in 30% sucrose overnight, cryostat sections were incubated in 2M HCl for 1 hr at 37°C to denature the DNA. BrdU was detected by immunohistochemistry with rat anti-BrdU (1:500, 347580, AbD Serotec, Hercules, California, USA, [Xu et al., 2010 (link)]). For tracing of neonatal brains, the tissue was fixed in PFA and DiI crystals (Molecular Probes, Eugene, Oregon, USA) were placed into either the visual or the somatosensory cortices using an insect needle. For anterograde tracing of TCAs at E16.5, brains were dissected along the midline and DiI crystals were placed in the dorsal thalamus. After incubation at 37°C for three weeks, the tissue was cut on a freezing microtome (30 μm, Leica, Nussloch, Germany), mounted on slides and stained with DAPI as described above.
6
Axonal Tracing via DiI Labeling
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For axonal tracing, small DiI crystals (1,1′-dioctadecyl 3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Molecular Probes) were inserted into the Th of hemi-dissected brains. These embryonic brains or cultured slices were incubated for 7 days, or 2 days, respectively, in 4% PFA at 37 °C. The brains were cut using a Leica vibratome (VT1000S) into 80–100 µm sections and counterstained with DAPI.
7
Multimodal Labeling of Organoids
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ALI-COs were visualised on an EVOS FL inverted microscope (ThermoFisher) and using a microinjection capillary <0.2 μl of 1 mg/ml AlexaFluor 647-conjugate CTB (ThermoFisher, C34778) or 1-4 DiI crystals (ThermoFisher, D3911) were applied to the target region. DiI tracing was performed multiple times but due to difficulty with dye uptake and low signal-to-noise ratio only in one experiment was tracing of sufficient quality for analyis. Similarly, to achieve sparse neuronal labeling, ALI-COs were injected with <0.2 μl of CytoTune emGFP Sendai fluorescence reporter (ThermoFisher, A16519). Four days after CTB and DiI labeling and 5 days after viral emGFP labelling the samples were fixed for histological analyses.
8
Spinal Cord Commissural Tracts Labeling
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Spinal cords from E12.5 murine embryos were dissected and mounted as open-books prior to fixation in 4% paraformaldehyde (PFA) for 18 hr. DiI crystals (D3911, ThermoFisher) were inserted in the most dorsal part of one hemi-spinal cord for anterograde labeling of commissural tracts. Axon trajectories were analyzed 24 hr later with a spinning disk microscope (Olympus X80). For each DiI crystal, a range of phenotypes can be observed. Classes were made representing the percentage of DiI crystals showing the phenotype over the total number of observed DiI crystals. Classes were assessed independently, with percentage ranging from 0% to 100%.
9
Multimodal Labeling of Organoids
10
Anterograde Tracing of Murine Commissural Axons
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Spinal cords from E12.5 murine embryos were dissected and mounted as open-books prior to fixation in 4% paraformaldehyde (PFA) for 18 hrs. DiI crystals (D3911, ThermoFisher) were inserted in the most dorsal part of one hemi-spinal cord for anterograde labeling of commissural tracts. Axon trajectories were analyzed 24 hrs later with a spinning disk microscope (Olympus X80). For each DiI crystal, a range of phenotypes can be observed. Classes were made representing the percentage of DiI crystals showing the phenotype over the total number of observed DiI crystals. Classes were assessed independently, with percentage ranging from 0% to 100%.
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