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Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage

Nature Protocols volume 1, pages 1977–1986 (2006)Cite this article

Abstract

In neuroscience, combining patch-clamping with protein identification in the same cell is becoming increasingly important to define which subtype or developmental stage of a neuron or glial cell is being recorded from, and to attribute measured membrane currents to expressed ion channels or receptors. Here, we describe a protocol to achieve this when studying cells in acute brain slices, which antibodies penetrate poorly into and for which detergent permeabilization cannot be used when using antibodies that recognize lipid components such as O4 sulfatide. The method avoids the need for resectioning of the electrophysiologically recorded slices. It employs filling of the cell with a fluorescent dye during whole-cell recording, to allow subsequent localization of the cell, followed by fixation and free-floating section labeling with up to three antibodies, which may recognize membrane, nuclear or cytosolic proteins. With practice, ∼80% of patch-clamped cells can be retrieved and have their proteins identified in this way. The entire protocol can be completed in 3–4 d.

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Figure 1: Documenting cell position in asymmetrical and symmetrical slices.
Figure 2: Combining whole-cell clamping with antibody labeling to GFAP.
Figure 3: Combining whole-cell clamping with antibody labeling to the lipid sulfatide O4.
Figure 4: Combining whole-cell clamping with antibody labeling to the proteoglycan NG2 and the nuclear transcription factor Olig2, and nuclear labeling with DAPI.
Figure 5: Combining whole-cell clamping with antibody labeling to the NMDA receptor subunit NR1.

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Acknowledgements

We thank D. Rowitch, C.D. Stiles & J. Alberta for Olig2 antibody; W. Stallcup for NG2 antibody; F.A. Stephenson, R.J. Wenthold & O.P. Ottersen for NR1 antibody; and W. Andrews, M. Catsicas, I. Hans, K. Jessen, R. Mirsky, P. Mobbs, S. Rakic and W. Richardson for advice. Funded by the Wellcome Trust grant number 075232.

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  1. Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK

    Ragnhildur Káradóttir & David Attwell

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  1. Ragnhildur Káradóttir
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Correspondence to Ragnhildur Káradóttir.

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Káradóttir, R., Attwell, D. Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage. Nat Protoc 1, 1977–1986 (2006). https://doi.org/10.1038/nprot.2006.261

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