Pipette solution for voltage-clamp recordings
0.5 mm EGTA
When using ion-sensitive indicator dyes such as fura-2, no additional substances that buffer the ion under study (i.e., EGTA or BAPTA) should be included in the pipette solution. The competition between the indicator dye and the additional buffer would seriously complicate the interpretation of the measurements.
10 mm HEPES
4 mm Mg-ATP
0.5 mm MgCl2
0.4 mm Na3-GTP
Adjust pH to 7.3 with CsOH.
The dye used will depend on the specifics of the experiment. 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.
Use highly purified water (e.g., W3500 from Sigma) for dissolving the dyes and for preparing the pipette solutions. Trace amounts of heavy metals or other contaminants may severely affect the fluorescence properties of the fluorophores.
Once prepared, dye-containing pipette solutions should be stored at −20°C until use. Fresh solutions should be prepared every 1-2 wk. For efficient use of expensive dyes with different pipette solutions, it is advisable to prepare appropriately concentrated (e.g., 125%) dye-free pipette solutions and a separate stock solution of the dye that will yield the desired concentration after dilution. Mix and sonicate stock solutions just prior to use. Dissolved in purified water, most dyes can be stored at −20°C for several months.
