[Neuroscience] Re: Biocytin fills?

[Imre Vida]

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