Protocol Note - Perforated patch
Today I could not find transfected neurons usable for imaging experiments. Instead, I decided to practice the perforated patch technique. One advantage of the perforated patch is that the technique is free from the so-called washout problem. Usually, patching to a cell, which makes a huge hole at the cell membrane, causes dialysis of cell components, and perturb some cell signalings. In the perforated patch, because the electric communication is through a small hole made of antibotics included in the patch pipette, it is thought that there is little washout of proteins or other biochemical compounds.
For now, in my hands, the technique does not work well for voltage clamp due to high seriese resistance, but is good enough for current-clamp measurements and eliciting action potentials. Followings are the current protocol I use. Further optimization is required.
Fresh Amphotericin B is solved in DMSO (f. 60mg/ml). The Amphotericin B stock is further diluted in a patch solution containing KCl 140mM, HEPES 10mM to 0.25 mg/ml, and filter with 0.22um pore. Front-fill the patch pipette (3-5MOhm) with the patch solution without Amphotericin B, and then back-fill with the patch solution with Amphotericin B.
After the formation of giga-ohm seal, seriese resistance decreased to ~200mOhm within 5 min, and to ~100MOhm within 30min.
