> Bill wrote:
> > Couple of quesions: When you say form an outside-out patch, do you
> > simpley mean withdraw the electrode from the cell? Is there any
> > particular method to this? Fast, slow etc...?
As Matt wrote you should withdraw the electrode slowly. Because
the membrane adheres very strong to the pipette, it follows
the pipette and will first form a "pipe" that still connects the
pipette and the cell but the access/series resistance increases
to several 10s to 100 MOhm range. At this stage you can reduce the holding V
to ~-40 mV and continue to withdraw the pipette slowly until
the resistance is in the GOhm range, as the 'pipe' gets more and more
narrow and at a certain point the two ends will be separated and resealed,
forming an outside-out patch on the pipette. A sudden drop in resistance
indicates that the pipe is ruptured, the cell is likely have a big hole.
Immediate fixation may still yield reasonable morphology in this case.
PFA: you should make it up in 0.1 M PB.