In 1949 Ling and Gerard used capillary glass tubes to make microelectrodes so that one could record the electrical activity of individual nerve cells before labeling them for staining, obtaining images of nerve cells that resembled the results of the Golgi method of staining (NicholsonandKater1973).
Modern Neuroscience Research Techniques
Author(s): U. Windhorst & H. Johansson Translated by Zhao Zhiqi Chen Jun
Operation method
Intracellular labeling assay of single Purkmje cells with horseradish peroxidase
Principle
The techniques and results used in this example are quoted from Bishop and King (1982), where a more detailed guide to the techniques can also be found (see also Kitai and Bishop 1981).
Materials and Instruments
Cerebellum in cats Move microelectrode Diameter 0.4-0.9/xm, conical shape, electrode inner liquid is 0.5mol/LKCl-Tris buffer containing 4%~10% HRP, pH 7.6, electrical impedance is 35~60Mfl. 3~5 depolarizing DC pulses per second, each energized for 100?200ms, current intensity 3~5nA, duration 3~5 min. Depending on the size of the nerve cells and the extent of filling, the animals can be immobilized 15 min to 30 h after HRP injection. First, 0.9% saline containing 2% lidocaine at 40 mg/kg was used for transvascular perfusion, and then Karnovsky-type fixative (sodium phosphate buffer containing 1% paraformaldehyde, 2% glutaric acid, and 2.5% glucose, pH 7.3, and a final concentration of 0.1 md/L) was used for perfusion. Serial cryosections (light microscopy) or vibrating sections (electron microscopy or light microscopy), slices were 50-60 pm thick, and sections were collected in phosphate buffer; DAB reaction; and sections for light microscopy were affixed with gelatin to chrome iso-coated slides (restained with cresyl violet). Electron or light microscopy sections were first dehydrated with acetone and then embedded with epoxy resin. Common Problems in the end The cytosol and dendrites of Purkinje cells have a very similar morphology after Golgi staining and intracellular labeling with HRP, except that the tiniest branches (dendritic spines) are well displayed when labeled with HRP [at least in a motor neuron, and the work of Brown and Fyffe (1981) suggests that intracellular labeling with HRP reveals a more complete picture of neuronal structures than any previous method]. that intracellular labeling with HRP provides a more complete picture of the dendritic structure of nerve cells than any previous method]. However, intracellular labeling with HRP is capable of labeling axons as well as lateral and terminal branches of axons (Fig. 1-6), whereas the Golgi method stains only the unmyelinated initial portion of the axon (Fig. 1-5B). In addition to its many functional applications, the HRP method can be used to study the fine structure and organization of cortico-nucleolar projections. For more product details, please visit Aladdin Scientific website.
Lidocaine Karnovsky-type fixative Cresyl violet
Microelectrodes Electron microscopy or light microscopy
