Combined Methods in Spinal Cord Reseach

Filling cells with biocytin during the patch-clamp recording

The whole-cell patch-clamp method provides important physiological data about the patched neuron, but this method applied alone does not say anything about the morphological properties of the neuron. If cells are filled with biocytin during the recordings, their morphological investigation becomes available.

As reported by Eckert, Willcockson and Light biocytin in higher concentrations may interfere with different sodium channels. For this reason only 0.1% (m/m) biocytin is added to the pipette filling solution. 10-15 minutes are more than enough to get perfect staining even when the biocytin is used in such low concentration. Following the neurophysiological recordings slices are fixed in 4% paraformaldehide for at least a couple of days. We found that biocytin remains in the cells for a few months even when the slices are stored in the fixative at 4C. Slices are resectioned and 50µm thick sections are cut on a vibratome. Fluorochrom labelled streptavidin is responsible for the visualization and identification of the patched cell. Further 2-3 antibodies can be applied simultaneously, shortly a conventional multiple immunofluorescent labelling is carried out and confocal microscopy and imaging softwares are used for the analysis.

Taken together biocytin is the "key-molecule" in this combination of methods, as it makes possible the morphological, immunohistochemical investigation of  patched and physiologically studied cells.



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