Intracellular injections were performed as described previously (Pérez de Sevilla Müller et al., 2007 (link), Müller et al., 2010a (link), Müller et al., 2010b (link)). tdTomato-expressing cells were visualized with a Zeiss 40X water-immersion objective. Borosilicate glass electrodes (#60200; A-M Systems; Sequim, WA) were pulled and filled at their tips with 0.5% Lucifer Yellow (Sigma-Aldrich) 4% N-(2-aminoethyl)-biotinamide hydrochloride (Neurobiotin; Vector Laboratories, Burlingame, CA), and back-filled with 0.1 M Tris buffer, pH 7.4. Under visual guidance provided by the tdTomato fluorescence, cells were targeted for injection. First, Lucifer Yellow was iontophoresed (−1 nA) into the labeled cells and when its morphology could be visualized, the polarity of the current was reversed (+1 nA) and Neurobiotin was injected for 3 minutes. After the final injection, the retina was kept in the bath solution for at least 30 minutes to allow diffusion of the Neurobiotin. The retinas were fixed in 4% PFA for 10 minutes. Neurobiotin was visualized by incubating injected retinas overnight at 4°C with streptavidin–FITC (1:500; Jackson ImmunoResearch, West Grove, PA) in 0.1 M PB containing 0.3% Triton X-100 (Sigma-Aldrich). Retinas were washed in PB 3 times for a total of 30 minutes and mounted in Vectashield (Vector Laboratories).
N 2 aminoethyl biotinamide hydrochloride neurobiotin
Manufactured by Vector Laboratories
Sourced in United States
N-(2-aminoethyl)-biotinamide hydrochloride (Neurobiotin) is a chemical compound used in biological research. It functions as a tracer molecule, allowing the visualization and tracking of neuronal connections and pathways.
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3 protocols using n 2 aminoethyl biotinamide hydrochloride neurobiotin
1
Intracellular Labeling of Retinal Cells
2
Intracellular Injection Method for Amacrine Cells
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Intracellular injections were performed as described previously (Pérez de Sevilla Müller et al., 2007 (link), 2010a (link),b (link); Vuong et al., 2015 (link)). Borosilicate glass electrodes (#60200; A-M Systems; Sequim, WA, USA) were pulled and filled at their tips with 0.5% Lucifer Yellow (Sigma–Aldrich) 4% N-(2-aminoethyl)-biotinamide hydrochloride (Neurobiotin; Vector Laboratories, Burlingame, CA, USA), and back-filled with 0.1 M Tris buffer, pH 7.4. In retinal whole mounts, amacrine cell bodies located in the proximal INL at the border of the IPL were targeted for injection. Lucifer Yellow was iontophoresed (−1 nA) into a single cell body and when the bistratified morphology of the AII amacrine cell was recognized, the polarity of the current was reversed (+1 nA) and Neurobiotin was injected for 3 min. The retinas were then fixed in 4% PFA for 10 min and washed for 30 min in 0.1 M PB. Neurobiotin was visualized by incubating the retinas with the injected cells overnight at 4°C with streptavidin–FITC (1:500; Jackson ImmunoResearch, West Grove, PA, USA) in 0.1M PB containing 0.3% Triton X-100 (Sigma–Aldrich). Retinas were washed in PB three times for a total of 30 min. The retinas were subsequently processed for immunohistochemical staining.
3
Whole-cell Electrophysiology of Spinal Motoneurons
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Spinal cord slices were prepared from P7–P9 rat cervical cords13 (link). Briefly, transverse slices (450–600 μm thick) of C6–C8 cervical segments were cut in oxygenated ice-cold cutting solution using a Linearslicer (PRO7, Dosaka EM, Kyoto, Japan) and transferred to a slice chamber perfused for more than 1 h at 15 ml/min with oxygenated artificial cerebrospinal fluid (ACSF).
Whole-cell currents were recorded from fluorescent MNs using patch-pipettes13 (link) (Fig.5 A). The internal solution in the pipettes contained 50 μM N-(2-aminoethyl) biotinamide hydrochloride (Neurobiotin, Vector Laboratories, Burlingame, CA, USA), which was later visualized within MNs by reacting it with Texas Red-avidin (Fig. 5 B). The whole-cell configuration was maintained for at least 20–40 min, which allowed the Neurobiotin to diffuse through the entire dendritic tree16 (link). Some of the morphological data presented in this study was obtained from common materials in our previous electrophysiological study13 (link).
Whole-cell currents were recorded from fluorescent MNs using patch-pipettes13 (link) (Fig.
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