Neurotrace 640 660, by Thermo Fisher Scientific - Product details

1

Visualizing Tetrode Implant Locations

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To enable determination of tetrode locations, after the last recording day we anaesthetised mice using an isoflurane chamber and pentobarbital (100–150 μl) and applied a 2-s ~20 µA current to burn the tissue at the tip of the electrodes. We then intracardially perfused phosphate-buffered saline (PBS, Gibco, 70011044, 10 times diluted with distilled water) for 2 min, then 4% paraformaldehyde (PFA, Sigma Aldrich, 30525-89-4) in 0.1 M phosphate buffer (PB, Sigma Aldrich, P7994) for 4 min at a 10 ml/min flow rate. We left the brains in 4% PFA in 0.1 M PB for 16 h, then transferred them to 30% sucrose (Sigma Aldrich, S0389) in PBS until they sank.
We cut 50 µm sagittal sections of the fixed brains using a freezing microtome. Sections were processed to label them with primary antibody rat anti-mCherry (Invitrogen M11217, 1:1000) followed by secondary antibody goat anti-rat Alexa 555 (Invitrogen A-21434, 1:1000) and stained with either NeuroTrace 640/660 (Invitrogen N21483, 1:500) or NeuroTrace 435/455 (Invitrogen N21479, 1:500) following procedures described previously^{43 (link)}. Images were taken on a Zeiss Axio Scan Z1 using a ×10 objective and visually inspected to determine the final position of the recording electrodes (see Supplementary Note 1).

Gerlei K., Passlack J., Hawes I., Vandrey B., Stevens H., Papastathopoulos I, & Nolan M.F. (2020). Grid cells are modulated by local head direction. Nature Communications, 11, 4228.

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2

Quantifying Cortical Neuron Expression

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To confirm widespread expression of TeLC in PCx, after recordings, mice were perfused with PBS followed by PFA (4%) and the brains were postfixed overnight. Coronal sections (50 μm) were taken through the A-P extent of PCx and permeabilized with Triton (0.1%). Brains were incubated overnight with a primary GFP antibody (Chicken Anti-GFP, Abcam, ab13970,1:500) and then washed and stained overnight with a secondary antibody (Goat Anti-Chicken Alexa Fluor 488, Abcam, ab150169; 1:500) and counter stained (NeuroTrace 640/660, Invitrogen, N21483; 1:400). Slices were mounted and imaged on an upright Zeiss 780 confocal microscope. Quantitative analyses were performed using ImageJ.

Bolding K.A, & Franks K.M. (2018). Recurrent cortical circuits implement concentration-invariant odor coding. Science (New York, N.Y.), 361(6407), eaat6904.

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3

Perfusion and Immunohistochemistry of Mouse Brains

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For anatomical experiments, animals were sacrificed 3–8 weeks after viral injections. Mice were administered a lethal dose of sodium pentobarbital and transcardially perfused with cold phosphate buffered saline (PBS) followed by cold paraformaldehyde (PFA, 4%). For immunohistochemistry in slices, brains were fixed for minimum for 24 hr in PFA at 4 °C, washed with PBS, and transferred to a 30% sucrose solution prepared in PBS for a minimum for 48 hr at 4 °C. Brains were then sectioned horizontally at 50–60 µm on a freezing microtome. To counterstain neurons, slices were incubated in a Neurotrace 640/660 (Invitrogen, Cat # N21483, 1:500) solution prepared in 0.3% PBS-T (Triton) for 24 hr at room temperature. Slices were washed with 0.3% PBS-T 3 x for 20 min and then mounted and cover-slipped with Mowiol. Mounted sections were stored at 4 °C.

Vandrey B., Armstrong J., Brown C.M., Garden D.L, & Nolan M.F. (2022). Fan cells in lateral entorhinal cortex directly influence medial entorhinal cortex through synaptic connections in layer 1. eLife, 11, e83008.

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4

Visualizing UBE3A Expression in Brain Sections

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Free-floating sections were incubated in 10% normal donkey serum (NDS), and then incubated overnight in a mouse monoclonal anti-UBE3A antibody (UBE3A, 1:10,000, Sigma-Aldrich Cat# SAB1404508, RRID:AB_10740376). Antigenic sites were visualized with donkey IgG conjugated to DyLight 549 (1:200, Jackson ImmunoResearch; West Grove, PA). Some sections were then counterstained with DAPI (1:10,000, Sigma-Aldrich, Cat# D9542) to visualize nuclei, and with NeuroTrace 640–660 (1:1000, Invitrogen, Thermo Fisher Scientific, Rockford, IL; Cat# N21483) to visualize neuronal somata. To verify antibody specificity, we performed immunocytochemistry on brain sections from AS model and UBE3A KO mice, run together with sections from WT mice. Experiments in which the primary antibodies were omitted were performed to control for nonspecific binding of the secondary antibody. Sections were examined with a Zeiss LSM 880 confocal microscope.

Burette A.C., Judson M.C., Burette S., Phend K.D., Philpot B.D, & Weinberg R.J. (2016). The subcellular organization of UBE3A in neurons. The Journal of comparative neurology, 525(2), 233-251.

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5

Mapping and Quantifying Oxytocin Neuron Projections

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The sections were rinsed with PBS-Tr (PBS with 0.1% Triton X-100; T8787, Sigma-Aldrich, UK) three times and then incubated in 1% blocking reagent (11096176001, Roche) diluted with PBS for at least 2 h, at room temperature.
For mapping the projections of oxytocin neurons, 60 µm–thick sections were incubated with rabbit anti-GFP antibody (1: 2000; ab13970) at 4 °C, overnight, followed by incubation with CF 568 goat anti-rabbit secondary antibody (20102; Biotium, Fremont, CA, USA), at room temperature, for 2 h. After the section was rinsed with PBS-Tr, it was reacted with NeuroTrace 640/660 (N21483, Invitrogen) and then incubated with 1 µg/mL DAPI for 20 min for nucleus targeting.
For cell counting, 45 µm–thick sections were incubated with rabbit anti-GFP antibody (1: 2000; ab13970) at 4 °C, overnight, followed by incubation with CF 488 goat anti-rabbit secondary antibody (20012; Biotium), at room temperature, for 2 h. After the section was rinsed with PBS-Tr, it was reacted with NeuroTrace 530/615 (N21482, Invitrogen); then cell counting was conducted.

Chuang H.J., Chang C.Y., Ho H.P, & Chou M.Y. (2021). Oxytocin Signaling Acts as a Marker for Environmental Stressors in Zebrafish. International Journal of Molecular Sciences, 22(14), 7459.

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6

Brain Perfusion and Imaging Protocol

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Deeply anesthetized mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) and brains were postfixed for 4 h at 4°C. 50 μm sections were cut on a vibratome, stained with fluorescent Nissl stain (NeuroTrace 640/660; Molecular Probes), and mounted in ProLong Antifade Diamond reagent (Invitrogen). Images were taken first with a slide-scanning wide-field microscope (VS120, Olympus), and high-resolution images of regions of interest were subsequently acquired with a Leica LS8 confocal microscope (Harvard NeuroDiscovery Center). Confocal images represent maximum intensity projections of 15 to 40 μm image stacks.

Piatkevich K.D., Jung E.E., Straub C., Linghu C., Park D., Suk H.J., Hochbaum D.R., Goodwin D., Pnevmatikakis E., Pak N., Kawashima T., Yang C.T., Rhoades J.L., Shemesh O., Asano S., Yoon Y.G., Freifeld L., Saulnier J.L., Riegler C., Engert F., Hughes T., Drobizhev M., Szabo B., Ahrens M.B., Flavell S.W., Sabatini B.L, & Boyden E.S. (2018). A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nature chemical biology, 14(4), 352-360.

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7

Brain Perfusion and Imaging Protocol

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Deeply anesthetized mice were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) and brains were postfixed for 4 h at 4°C. 50 μm sections were cut on a vibratome, stained with fluorescent Nissl stain (NeuroTrace 640/660; Molecular Probes), and mounted in ProLong Antifade Diamond reagent (Invitrogen). Images were taken first with a slide-scanning wide-field microscope (VS120, Olympus), and high-resolution images of regions of interest were subsequently acquired with a Leica LS8 confocal microscope (Harvard NeuroDiscovery Center). Confocal images represent maximum intensity projections of 15 to 40 μm image stacks.

Piatkevich K.D., Jung E.E., Straub C., Linghu C., Park D., Suk H.J., Hochbaum D.R., Goodwin D., Pnevmatikakis E., Pak N., Kawashima T., Yang C.T., Rhoades J.L., Shemesh O., Asano S., Yoon Y.G., Freifeld L., Saulnier J.L., Riegler C., Engert F., Hughes T., Drobizhev M., Szabo B., Ahrens M.B., Flavell S.W., Sabatini B.L, & Boyden E.S. (2018). A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nature chemical biology, 14(4), 352-360.

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8

Immunostaining of Mouse Brain Sections

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Mice were administered ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) and euthanized by transcardial perfusion with 10 mL of PBS, followed by 10 mL of 4% paraformaldehyde in PBS. Brains were extracted and 100 μm coronal sections were cut on a vibratome. The tissue was labeled with the following antibodies: goat anti-GFP (Abcam ab6673), rabbit anti-arc (Synaptic Systems 156-003), goat anti-c-fos (Santa Cruz sc-52-G), alexa-488 donkey anti-goat (Jackson ImmunoResearch), alexa-568 donkey anti-rabbit (Life Technologies A11057), alexa-647 donkey anti-rabbit (Jackson ImmunoResearch 711-605-152) and alexa-568 donkey anti-goat (Life Technologies A10042). Slices were counterstained with neurotrace 640/660 or 435/455 (Life Technologies N21483 or N21479, respectively). Antibody amplification was not used to visualize eNpHR3.0-eYFP. All images were taken using a Zeiss LSM-710 confocal microscope system.

Root C.M., Denny C.A., Hen R, & Axel R. (2014). The participation of cortical amygdala in innate, odor-driven behavior. Nature, 515(7526), 269-273.

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9

Immunostaining of Mouse Brain Sections

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Mice were administered ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively) and euthanized by transcardial perfusion with 10 mL of PBS, followed by 10 mL of 4% paraformaldehyde in PBS. Brains were extracted and 100 μm coronal sections were cut on a vibratome. The tissue was labeled with the following antibodies: goat anti-GFP (Abcam ab6673), rabbit anti-arc (Synaptic Systems 156-003), goat anti-c-fos (Santa Cruz sc-52-G), alexa-488 donkey anti-goat (Jackson ImmunoResearch), alexa-568 donkey anti-rabbit (Life Technologies A11057), alexa-647 donkey anti-rabbit (Jackson ImmunoResearch 711-605-152) and alexa-568 donkey anti-goat (Life Technologies A10042). Slices were counterstained with neurotrace 640/660 or 435/455 (Life Technologies N21483 or N21479, respectively). Antibody amplification was not used to visualize eNpHR3.0-eYFP. All images were taken using a Zeiss LSM-710 confocal microscope system.

Root C.M., Denny C.A., Hen R, & Axel R. (2014). The participation of cortical amygdala in innate, odor-driven behavior. Nature, 515(7526), 269-273.

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10

Immunostaining of Quail Neural Cultures

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Quail cultures (grown directly on plastic plates) were fixed by 4% paraformaldehyde (PFA) for 15 min, permeabilized with PBS containing 5% FBS (fetal bovine serum) and 0.1% triton X-100. NeuN or MAP2-immunostaining were performed by overnight incubation of fixed cells with anti-NeuN antibody (mouse, clone A60, Millipore CAT# MAB377) (1:1000 in PBS) or with anti-MAP2 (mouse, cat # MA5-12826, Thermo Fisher) (1:500 in PBS) at 4 °C, with 3% FBS and 0.1% triton X-100. The next day, plates were washed three times with PBS and stained by secondary antibody (Rhodamine Red-X goat Anti-mouse IgG, Jackson Laboratories, CAT# 115-295-003) (1:200), in 3% FBS and 0.1% triton X-100 for 1 h at room temperature. Plates were then washed by PBS and stained with DAPI (1:1000). NeuroTrace 640/660 staining (Thermo Fisher) was performed as previously described in ref. ^{122 (link)}. Briefly, cultures were fixed as described above, permeabilized with PBS containing 0.1% triton X-100 for 10 min, washed, and followed by incubation for 30 min with PBS containing NeuroTrace stain (1:100). Then, cells were washed by PBS and stained with DAPI (1:1000). GFAP-staining (1:500) was performed by using anti-GFAP (mouse, clone G-A-5, Calbiochem, CAT# IF03L), followed by staining with a secondary antibody (1:500) (Alexa Fluor-488 goat anti-mouse IgG, Jackson Laboratories, CAT# 115-545-003).

Zoabi S., Andreyanov M., Heinrich R., Ron S., Carmi I., Gutfreund Y, & Berlin S. (2023). A custom-made AAV1 variant (AAV1-T593K) enables efficient transduction of Japanese quail neurons in vitro and in vivo. Communications Biology, 6, 337.

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Neurotrace 640 660

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19 protocols using neurotrace 640 660

Visualizing Tetrode Implant Locations

Quantifying Cortical Neuron Expression

Perfusion and Immunohistochemistry of Mouse Brains

Visualizing UBE3A Expression in Brain Sections

Mapping and Quantifying Oxytocin Neuron Projections

Brain Perfusion and Imaging Protocol

Brain Perfusion and Imaging Protocol

Immunostaining of Mouse Brain Sections

Immunostaining of Mouse Brain Sections

Immunostaining of Quail Neural Cultures

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