Thick wall capillary glass and access resistance

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Recorder
Recorder's picture
Thick wall capillary glass and access resistance

I have two questions. Thank you for having an interest in my question and please give me some advice.

Question 1>
I have a trouble for making patch pipette with thick wall capillary glass. I've have been used thin wall 1mm O.D. capillary glass, and it was easy to make appropriate shape and diameter with them.
To change the capillary glass from thin wall to thick wall, I struggled to adjust my pipette puller and finally I made a good example. But every time I make a new pipette with same setting the puller make quite different diameter of pipettes. It is not consistent. The pipette puller that I used was Narishige vertical type puller.

I tried to do same thing with Sutter horizontal puller, and I also found good setting values.. But it is also not consistent.

Does this just mean that I'm using wrong setting values and that I have to find more apt setting values?
Are there anyone who experience similar problem with thick wall capillary glass?

Question 2>

I'm doing slice whole cell patch clamp recording experiment with 2 or 3 weeks rat brain. It seems that getting whole cell configuration is not very difficult now, but I have a terrible problem with access resistance change during the whole cell configuration experiment. I think I usually throw almost 70% of results away due to the Ra change during the recording. My experiment is not even very long. The whole experiment protocol takes just 10 min.

I want to know whether this level of Ra problem is typical.

And, can changing pipette make it better? Are there anything that I can do to stabilize the Ra other than perforated patch?

Thanks a lot.

JennaMo
JennaMo's picture
I have experience with the

I have experience with the Sutter puller. To create a stable program you need to be in the middle of the velocity range for the number of loops it takes to pull your glass. You can do this by keeping everything else the same and looking for the range of velocities which keep you within, say, 4 loops. They outline how to do this in the manual for the puller.

I find that for thick-walled glass 4 loops is ideal to create the resistance and shape that I need. You need to tailer each line of the program for what you want. This can be quite time consuming but worth the effort once you get a good, stable program. I recomend the manual for the puller, the Sutter Pipette Cookbook and a chapter in Current Protocols in Neuroscience for advice on puller programs.

Hope this is of some help.

I can't help too much with your question 2. I have similar issues with my whole cell recording in a mouse brain slice. You can try appliying a liitle positive pressure to re-open the seal when the Ra increases, but this hasn't helped much for me.

Jenna.

The FFM
The FFM's picture
 RE Q1

 RE Q1

I recommend downloading Sutter's Pipette Cookbook (click the link) to learn how to better optinmize your pipette shape on a horizontal puller using different glass types.

remember you do a ramp test every day to make sure your program heat setting is working withing the parameters of the condition of the filament in the puller.

RE Q2

once you've got the hang of adjusting your puller programs you can develop one that pulls a more triangular shape tip (i.e. small tip with rapidly widening glass behind it) rather than one that curves slowly to a small tip with a longer thinner shaft.
 
Although the evidence is not conclusive, membrane resealing is thought to be somewhat Ca2+ dependent, so if your experiment type allows, you may also consider experimenting with the concentration of EGTA in the intracellular solution.