Thanks guys,
That might explain why I had no staining, I was withdrawing the
electrode as fast as possible (2mm a second).
Thanks again.
Imre Vida wrote:
> Hi Bill,
>> > 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...?
>> Outside-out patch:
> 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.
>>> Cheers
>> imre
