> 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...?
(Imre, please forgive me for answering a question directed to you.
Please correct anything you think needs correcting. And thanks for the
biocytin recipes! - we'll try 'em out soon)
For outside-out patches, safest thing is probably to go pretty slow. In
a slice, when we deliberately want outside-outs that we can record
single channel activity from, we usually withdraw as slow as
manipulators will allow.
You will know that you have an outside-out patch when:
1) You originally broke in normally and got a healthy whole-cell
recording with good series resisitance (<5-10 MOhm).
2) When you finally withdraw completely, things will go back to how
they looked when you inittially made the SEAL (e.g., very sharp
capacitive transients, reflecting pipette capacitance only rather than
whole-cell capacitance, and you should also get back to ~GOhm seal
resistance.) So now instead of an on-cell seal you have a tightly
sealed outside-out patch. Get it?
3) The point of this is to make sure that when you withdraw, you don't
leave a giant bleeding hole in the cell, or rip the cell body out of
the slice. When you form an outside-out patch, one end of the membrane
tube you are pulling has sealed up onto the patch pipette, and the
assumption is that the other end of it will have sealed back up on the
cell (thus not leaving a giant bleeding hole). Finally, sometimes you
can get a big blob on the end of your pipette, which is probably a bad
sign meaning that you pulled the nucleus and part of the cell body up
with you (i.e., a nucleated-patch). On a good outside-out patch, you
won't be able to see anything hanging off your pipette, it'll probably
look like just an empty pipette tip.
Note: in a slice you often have to withdraw a loooooong way (possibly
hundreds of microns - no kidding) before the patch finally separates
from the cell and seals up. In culture, for some reason, you rarely
need to withdraw that far.
By the way, we've tried the ascorbate/pyruvate slicing solution that
you recommended a while back, and we're routinely using it now. It
definitely did change the look of our tissue for the better, and also
helped a bit with resting potentials being more negative. This was in
dentate granule cells.