At 11 wpl, a second complete spinal cord transection was performed 5 mm below the site of the original transection and the retrograde tracer Neurobiotin (Vector) was applied in the rostral end of the transected spinal cord with the aid of a minute pin (#000). The animals were allowed to recover at 19°C with appropriate ventilation conditions for 1 week to allow transport of the tracer from the application point to the neuronal soma of descending neurons (the M1, M2, M3, I1, I2, I3, I4, I5, I6, B1, B2, B3, B4, B5, B6, Mauthner (Mth) and auxiliary Mauthner (Mth′) neurons were analyzed; Fig. S3 ). Since the original SCI also was a complete spinal cord transection, only neurons whose axons regenerated at least 5 mm below the site of injury were labelled by the tracer. Brains of these larvae were dissected out, the posterior and cerebrotectal commissures of the brain were cut along the dorsal midline, and the alar plates were deflected laterally and pinned flat to a small strip of Sylgard (Dow Corning Co., USA) and fixed with 4% PFA in TBS for 4 h at 4°C. After washes in TBS, the brains were incubated at 4°C with Avidin D-FITC conjugated (Vector; Cat#: A-2001; 1:500) diluted in TBS containing 0.3% Triton X-100 for 2 days to reveal the presence of Neurobiotin. Brains were rinsed in TBS and distilled water and mounted with Mowiol.
Neurobiotin
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Canada
Neurobiotin is a water-soluble, low molecular weight tracer molecule used for the visualization and tracing of neuronal connections in the nervous system. It is a non-toxic, non-radioactive compound that can be detected using histochemical methods.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (4 mentions)
Pli 188, by Nikon (4 mentions)
Mowiol, by Merck Group (3 mentions)
Ames medium, by Merck Group (3 mentions)
C1si fast spectral confocal system, by Nikon (3 mentions)
Tetramethyl rhodamine labeled dextran, by Thermo Fisher Scientific (3 mentions)
Lucifer yellow, by Vector Laboratories (3 mentions)
Cy3 conjugated streptavidin, by Jackson ImmunoResearch (3 mentions)
Lucifer yellow, by Merck Group (3 mentions)
Streptavidin alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Abc reagent, by Vector Laboratories (2 mentions)
Sulforhodamine b, by Merck Group (2 mentions)
Anti gfp a 11122, by Thermo Fisher Scientific (2 mentions)
Vectabond, by Vector Laboratories (2 mentions)
Streptavidin cy2 conjugate, by Jackson ImmunoResearch (2 mentions)
Alexa 594, by Thermo Fisher Scientific (2 mentions)
Streptavidin cy2, by Jackson ImmunoResearch (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Neurobiotin, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
91 protocols using neurobiotin
1
Retrograde Labeling of Spinal Cord Neurons
2
Neurobiotin Labeling and Confocal Imaging of Neurons
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Neurobiotin (Vector Lab) was dissolved in a modified internal solution [70 mM K-gluconate, 6 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, 1 mM EGTA, 4 mM Mg-ATP, 0.5 mM GTP-tris, and 10 mM Hepes (pH 7.4)] for osmolarity balance of the 2% Neurobiotin. The recording pipette was filled with Neurobiotin-containing internal solution. After breaking into whole-cell mode, depolarizing currents (200 ms, 2 Hz) were injected for 20 min, which facilitated diffusion of positively charged Neurobiotin into the recorded neuron. The recording pipette was gently detached from the cell after another 20-min wait of Neurobiotin diffusion within the cell. The labellar preparation was transferred into 4% paraformaldehyde, fixed for 4 hours on ice, and then washed by phosphate-buffered saline (PBS) for three times at a 20-min interval, blocked in 5% bovine serum albumin in PBST (1% Triton X-100 in PBS) for 2 hours at room temperature. The preparations were incubated with Streptavidin-568 (1:500; Invitrogen, catalog number S11226) overnight at 4°C, washed by PBST (1% Triton X-100 in PBS) for three times at an interval of 20 min, and then mounted in the glycerol. Images were acquired on a Nikon A1R+ confocal microscope with a 25× water immersion objective.
3
Labeling Single Purkinje Cells in Mice
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We labelled Purkinje cells in anaesthetized 3–5-month-old mice using 8–10 MΩ glass electrodes that were filled with 1% neurobiotin (Vector Laboratories Inc., Burlingame, CA, USA) diluted in 0.9% sterile saline. After identifying and then entraining a single unit, we performed a loose-patch fill77 (link) by delivering 1–5 nA of positive current for 45–60 min. The mice were then perfused and the neurobiotin labelling of single filled cells detected using the VECTASTAIN Elite ABC method (Vector Laboratories Inc.) and visualized with DAB.
4
Tracing Descending Neurons in Larval Spinal Cord
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The origin of PmCRH‐ir fibers observed in the spinal cord was investigated by neuronal tract tracing combined with immunofluorescence. Tract‐tracing experiments were performed in larval samples to label descending neurons that innervate the spinal cord. Neurobiotin (MW 322.8 Da; Vector, Burlingame, CA, USA) was used as a tracer. The larval spinal cord was exposed by a longitudinal incision made in the dorsal region of the body at the level of the fifth gill and completely cut with Castroviejo scissors. The tracer was applied in the rostral stump of the transected spinal cord with the aid of a minute pin (#000). The animals were allowed to recover at 19.5°C with appropriate aeration conditions for 7 days to allow transport of the tracer from the application point to the neuronal soma of descending neurons. Brains of these larvae were fixed with 4% PFA and processed for PmCRH immunofluorescence as above. After the immunofluorescence protocol, the sections were incubated at room temperature with Avidin D‐FITC conjugated (dilution 1:1000; Vector; Cat#: A‐2001) diluted in TBS containing 0.3% Triton X‐100 for 4 h to reveal the presence of Neurobiotin. Slides were rinsed in TBS and distilled water and mounted with Mowiol® (Sigma).
5
Neurobiotin Labeling of Neurons
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iNGNs, grown exactly as for electrophysiology experiments, were filled with Neurobiotin (Vector Laboratories, Burlingame, USA) using a patch-clamp glass pipette containing 0.5% Neurobiotin in the pipette solution, for 20–45 minutes, and 200 ms depolarizing 500 pA current pulses at 1 Hz for 2 min. The pipette was very slowly raised from the cell body and then the coverslip was transferred to a 24-well plate and gently washed in PBS for 5 min before being fixed with 4% paraformaldehyde in PBS containing Mg2+and Ca2+ for 20 min. Coverslips were washed 3x with PBS and then incubated in Elite ABC reagent (Vector Laboratories) for 2 hr. After washing 3x with PBS, the coverslips were incubated in DAB peroxidase substrate solution (Vector Laboratories) for 10 min, followed by one wash with ddH2O. The coverslips were mounted with Aqua-Poly/Mount onto slides and stored upright at room temperature.
6
Neurobiotin Staining and Neuron Reconstruction
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Slices with cells filled with Neurobiotin (Vector Laboratories, Burlingame, CA) during current clamp experiments were fixed in 3% glutaraldehyde for a minimum of 24 hours. Slices were washed in 0.1 M phosphate buffer (PB) and incubated in 0.5% H2O2 for 30 minutes. Slices were then washed in PB and incubated in ABC reagent (Vector Laboratories, Burlingame, CA) containing Avidin DH and biotinylated horseradish peroxidase H for 24-48 hours at 4°C. Slices were then incubated in DAB solution (Vector Laboratories, Burlingaxme, CA) in the presence of H2O2 and monitored for a visible color change to the slices and Neurobiotin filled cell. Slices were dehydrated in glycerol and mounted on glass slides for imaging. Identifiable neurons were reconstructed at 40X magnification using a Leitz Diaplan microscope with Neurolucida 6.0 software (MicroBrightField, Inc., Williston, VT). The Sholl radius was set so that there were 10 and 25 concentric circles measuring the basal dendrites and apical dendrites respectively.
7
Tracing Descending Neurons Innervating Spinal Cord
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The origin of PmCCK-8-ir fibres observed in the spinal cord was investigated by neuronal tract tracing combined with immunofluorescence. Tract-tracing experiments were performed in larval samples to label descending neurons that innervate the spinal cord. Neurobiotin (NB, 322.8 Da molecular weight; Vector; Burlingame, CA) was used as a tracer. The larval spinal cord was exposed by a longitudinal incision made in the dorsal region of the body at the level of the fifth gill and completely cut with Castroviejo scissors. The tracer was applied in the rostral stump of the spinal cord with the aid of a minute pin (#000). The animals were allowed to recover at 19.5 ºC with appropriate aeration conditions for 7 days to allow transport of the tracer from the application point to the neuronal soma of descending neurons. Brains of these larvae were fixed with 4% PFA as above and processed for PmCCK-8 immunofluorescence.
Then, the sections were incubated at room temperature with Avidin D-FITC conjugated (dilution 1:1,000; Vector; Cat#: A-2001) diluted in TBS containing 0.3% Triton X-100 for 4 hours to reveal the presence of Neurobiotin. Slides were rinsed in TBS and distilled water and mounted with Mowiol (Sigma).
Then, the sections were incubated at room temperature with Avidin D-FITC conjugated (dilution 1:1,000; Vector; Cat#: A-2001) diluted in TBS containing 0.3% Triton X-100 for 4 hours to reveal the presence of Neurobiotin. Slides were rinsed in TBS and distilled water and mounted with Mowiol (Sigma).
8
Mapping Descending Neurons in Larval Spinal Cord
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The origin of PmCRH-ir fibers observed in the spinal cord was investigated by neuronal tract tracing combined with immunofluorescence. Tract-tracing experiments were performed in larval samples to label descending neurons that innervate the spinal cord. Neurobiotin (MW 322.8 Da; Vector; Burlingame, CA) was used as a tracer. The larval spinal cord was exposed by a longitudinal incision made in the dorsal region of the body at the level of the fifth gill and completely cut with Castroviejo scissors. The tracer was applied in the rostral stump of the transected spinal cord with the aid of a minute pin (#000). The animals were allowed to recover at 19.5 ºC with appropriate aeration conditions for 7 days to allow transport of the tracer from the application point to the neuronal soma of descending neurons. Brains of these larvae were fixed with 4% PFA and processed for PmCRH immunofluorescence as above. After the immunofluorescence protocol, the sections were incubated at room temperature with Avidin D-FITC conjugated (dilution 1:1000; Vector; Cat#: A-2001) diluted in TBS containing 0.3% Triton X-100 for 4 hours to reveal the presence of Neurobiotin. Slides were rinsed in TBS and distilled water and mounted with Mowiol® (Sigma).
9
Identifying Type-5 Bipolar Cells
10
Electrophysiological Recordings in Ames' Medium
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