Dye loading with patch pipettes.
This protocol describes the loading of individual cells with fluorescent probes via patch pipettes. The patch-clamp methodology has been successfully used for single-cell dye labeling in cultured neurons, brain slices, and in vivo preparations. A broad range of dyes can be used with this loading technique. Markers for morphological reconstruction (e.g., Lucifer yellow); ion-sensitive indicator dyes for monitoring second-messenger cascades (e.g., fura-2); and dye-labeled proteins for fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP) studies are all suitable for patch-clamp loading. The most widespread application of this technique has been for Ca(2+) imaging. Whole-cell patch-clamp recordings represent a versatile loading technique that allows combined electrophysiological and optical measurements at a quantitative level.
Jens
J
Eilers
Arthur
A
Konnerth
D000818:Animals; D001921:Brain / Q000473:pathology; D002118:Calcium / Q000737:chemistry; D004594:Electrophysiology / Q000295:instrumentation* / Q000379:methods; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D018408:Patch-Clamp Techniques / Q000295:instrumentation* / Q000379:methods; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D013194:Staining and Labeling / Q000295:instrumentation* / Q000379:methods
D005456:Fluorescent Dyes; D002118:Calcium