To verify final tetrode location we performed electrolytic lesions (100 μA, ~1.5 min per lead) after the last recording session. One day following lesion, mice were overdosed with an intraperitoneal injection of sodium pentobarbital (100 mg/kg) and transcardially perfused with saline followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). After perfusion, brains were submerged in 4% PFA in 0.1 M PB for 24 hours for post-fixation and then cryoprotected for 24 hours by immersion in 30% sucrose in 0.1 M PB. The brain was encased in the same sucrose solution, and frozen rapidly on dry ice. Serial coronal sections (60 μm) were cut on a sliding microtome for reconstruction of the lesion site and tetrode tracks. Fluorescent Nissl (NeuroTrace, Invitrogen) was used to identify cytoarchitectural features of the SC and verify tetrode tracks and lesion damage within or below the SC. Images of the SC were captured with a 10x objective lens, using a 3I Marianis inverted spinning disc confocal microscope (Zeiss). Arch expression and optical fiber depth were verified followed the same procedures with the exception of electrolytic lesioning, and allowed us to confirm that effective optical stimulation was limited to the intermediate and deep layers of the left SC.
Neurotrace
Manufactured by Thermo Fisher Scientific
Sourced in United States, Panama
NeuroTrace is a fluorescent dye used for staining neurons in fixed tissue samples. It binds to Nissl substance in the cytoplasm of neurons, allowing for the visualization and identification of neuronal cell bodies.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Pentobarbital sodium, by Merck Group (3 mentions)
Application suite advanced fluorescence software, by Leica (3 mentions)
Fluoromount g, by Southern Biotech (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Vt1000, by Leica (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Confocal laser scanning microscope 700, by Zeiss (2 mentions)
Confocal microscopy, by Leica (2 mentions)
Chicken anti gfp, by Abcam (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm780, by Vector Laboratories (2 mentions)
Mouse anti neun, by Merck Group (2 mentions)
Sm2010r, by Leica (2 mentions)
Prolong diamond, by Thermo Fisher Scientific (2 mentions)
Neurolucida 360, by MicroBrightField (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Rabbit anti substance p, by Immunostar (2 mentions)
62 protocols using neurotrace
1
Verification of Tetrode Placement in SC
2
Quantitative Analysis of DRG Neuron Transduction
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Different approaches for quantitative analysis of transduction in DRG neurons were applied for intrathecal and intracolonic AAV delivery to allow comparisons with previous literature. For intrathecal AAV delivery, DRG sections were immunolabeled and counterstained with NeuroTrace (1:1000, Invitrogen). Confocal images were collected with the Olympus Fluoview system as described above (UPLAPO 10x/0.4 NA), using uniform imaging parameters that avoided saturation. Image analysis was performed by trained observers blinded to the experimental groups using Fiji. NeuroTrace-labeled neuronal profiles with nuclei were outlined in 5 evenly spaced sections per DRG, and measurements of the area and the mean grey value of tdTomato immunoreactivity in each profile were obtained. To estimate the proportion of labeled neurons, a threshold mean gray value was selected for the entire data set based on the rate of change of the slope of the cumulative mean gray value frequency distribution.
3
Verifying Tetrode Placement in Mouse Brain
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To verify final tetrode location we performed electrolytic lesions (100 μA, ~1.5 min per lead) after the last recording session. One day following lesion, mice were overdosed with an intraperitoneal injection of sodium pentobarbital (100 mg/kg) and transcardially perfused with saline followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). After perfusion, brains were submerged in 4% PFA in 0.1 M PB for 24 hr for post-fixation and then cryoprotected for 24 hr by immersion in 30% sucrose in 0.1 M PB. The brain was encased in the same sucrose solution, and frozen rapidly on dry ice. Serial coronal sections (60 μm) were cut on a sliding microtome for reconstruction of the lesion site and tetrode tracks. Fluorescent Nissl (NeuroTrace, Invitrogen) was used to identify cytoarchitectural features of the SNr and verify tetrode tracks and lesion damage within or below the SNr. Images of SNr (see Figure 2A ) were captured with a 10x objective lens, using an LSM 5 Pascal series Axioskop 2 FS MOT confocal microscope (Zeiss).
4
Histological Verification of Implant Placement
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Final tetrode location, fiber, and cannula placement, and viral expression was confirmed histologically (Supplementary Fig. 1 ). Mice were overdosed with an intraperitoneal injection of sodium pentobarbital (100 mg/kg; Sigma Life Science) and transcardially perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). After perfusion, brains were submerged in 4% PFA in 0.1 M PB for 24 h for post-fixation and then cryoprotected for at least 12 h immersion in 30% sucrose in 0.1 M PB. On a freezing microtome, the brain was frozen rapidly with dry ice and embedded in 30% sucrose. Serial coronal sections (50 µm) were cut and stored in 0.1 M PBS. Sections were stained with 435/455 blue fluorescent Nissl (1:200, NeuroTrace; Invitrogen) to identify cytoarchitectural features of the SC and verify tetrode tracks and implant placement. Images of the SC were captured with a ×10 or ×20 objective lens, using a 3I Marianis inverted spinning disc confocal microscope (Zeiss) and 3I Slidebook 6.0 software. Images were adjusted in ImageJ to enhance contrast.
5
Immunohistochemical Analysis of Optogenetically-Targeted Neurons
6
Quantifying Anti-AQEE30 Immunoreactivity in Neuropathic Pain
7
Visualizing Neuronal Anatomy and Function
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Mice were anesthetized with pentobarbital and transcardially perfused with ice-cold 4% paraformaldehyde in phosphate buffer (PB). Brains were dissected, post-fixed for 24 hr at 4°C and cryoprotected with solution of 30% sucrose in 0.1M PB at 4°C for at least 24 hr, cut into 30 µm sections and processed for Nissl body staining. Sections were washed three times in PBS and blocked in PBS containing 0.5% Triton X-100 (G-Biosciences) for 1 hr. This was followed by a 1 hr incubation with fluorescent Nissl stain to allow visualization of cell bodies (1:400, Neurotrace, Invitrogen RRID:SCR_008410 ). Sections were then washed three times in PBS, followed by three 10 min rinses in PB and mounted on glass slides with Hard set Vectashield (Vector Labs) for episcope microscopy. Correct regional expression of the AAV5-DIO-ChR2-eYFP was verified in addition to placement of the opto-dialysis probe in either the vNAcSh or dNAcSh, which are represented on hit maps.
8
Viral Injection Site Visualization in Murine Brain
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Coronal hippocampal sections were cut right after amygdala sections, immediately imaged on a fluorescent stereoscope (SCX16, Olympus, Japan) to confirm viral injection sites, and slices were fixed in 4% PFA in PBS for further analyses. The frontal cortex was removed, fixed overnight in 4% PFA in PBS, resectioned at 70 μm, and stained with Neurotrace (1:200, Invitrogen). Bead only injection sites in the mPFC were imaged using a fluorescent stereoscope. Viral and bead injection sites in the mPFC were imaged on a laser scanning confocal microscope either with a 10 × 0.3° NA or a 25 × 0.8° NA objective with the pinhole open or set to one airy unit as indicated. All images were overlaid with the mouse brain atlas (Paxinos and Franklin, 2001 ).
9
Immunofluorescence Staining of DRG Neurons
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Thawed DRG sections were incubated in a blocking buffer (PBS containing 0.3% Triton-X 100, 1% BSA, 1% normal donkey serum) for 30 min followed by overnight incubation in primary antibody solution (Rabbit anti-DsRed (1:1,000), Living Colors®; Chicken anti-GFP(1:1,000), Abcam #13970) at 4°C. Following three 10-min washes in PBS, slides were incubated with secondary antibody solution (Cy3 Donkey anti-Rabbit 1:600, Alexa488 Donkey anti-Chicken 1:100, Jackson labs) for 1 h at RT, washed again with PBS (3 × 10 min), and stained with NeuroTrace (1:1,000, Invitrogen #N21479). Coverslips were placed onto the slides using FluorSave mounting media.
10
Immunohistochemical Staining of Mouse Brains
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