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

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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.