Yeah, so if you unscrew the pipette holder from the headstage you'll see a gold pin, and around that gold pin there is a plastic cap that can unscrew from the rest of the pipette holder (it usually is done up extremely tightly, I often have to resort to using my teeth to get it off). Check that this cap is screwed on tightly. If it isn't, tighten it up. If it's already tight, unscrew that cap (being careful of the metal side port). Underneath that cap you will see the electrode wire and a silicone seal. This is what is causing your leak. If you look in one of the pink bags that should be in the box that the headstage came in, you will find more of the silicone tube that the seal is made from. Cut off a 3mm length, this is your new seal. Get the electrode wire. Make sure that it has a nice clean end, and is bent into a little L (where it says 90 degree bend in the attached diagram). Thread the wire through the center of the seal, and put the seal and wire back in the pipette holder. Make sure that when you put the wire in, the bent section isn't so long it touches the barrel of the pipette holder. Then put the pipette holder back together.

Hi Tiziano,

I think your idea about a leak in your positive pressure is the best guess, and exactly what I thought when I read your description. Your series resistance should only go up by, at most, 50% when you approach a cell. It should never go up 300%.

You need to confirm that a) your system holds positive pressure and b) that positive pressure it getting to the tip of your electrode.

So, for a) When you lock on the positive pressure, and leave it for a minute, and then release the positive pressure, can you hear the "pop" as the pressure is released? For b) When your electrode approaches the slice you should be able to see the tissue change as the pipette solution is blown out.

Hi William,

Thanks for your answer, effectively I found out that the problem is due to my and your first guess, a leak in the support. Even though I already changed all the support the leak seems to come from the white ring that connects the pipette holder to the manipulator (see picture). Do you have any advise on how can I fix it (my boss is out for his holidays and if I can't fix it I can't go on with my experiments...)?

Best

Yeah, so if you unscrew the pipette holder from the headstage you'll see a gold pin, and around that gold pin there is a plastic cap that can unscrew from the rest of the pipette holder (it usually is done up extremely tightly, I often have to resort to using my teeth to get it off). Check that this cap is screwed on tightly. If it isn't, tighten it up. If it's already tight, unscrew that cap (being careful of the metal side port). Underneath that cap you will see the electrode wire and a silicone seal. This is what is causing your leak. If you look in one of the pink bags that should be in the box that the headstage came in, you will find more of the silicone tube that the seal is made from. Cut off a 3mm length, this is your new seal. Get the electrode wire. Make sure that it has a nice clean end, and is bent into a little L (where it says 90 degree bend in the attached diagram). Thread the wire through the center of the seal, and put the seal and wire back in the pipette holder. Make sure that when you put the wire in, the bent section isn't so long it touches the barrel of the pipette holder. Then put the pipette holder back together.

I agree that it's most likely a leak in the suction line. It can also be dirts or bubbles in your pipette solution that block the outflow and therefore increase the resistance. For the bubbles, make sure that there is not any by gently taping the pipette or shaking it before mounting it onto the holder. usually if you're not in the tissue, adding negative pressure should improve the resistance in that case. For the dirt in your internal solution you can try to filter it, it did help me with some internals.

Good luck

Hi Christophe, no I don't think that the bubbles and the debris are not the issue, I am already using a filter and I'm always shaking the pipette before using it, making sure that all the bubbles from the top.

Hi Linda,

thanks for the advise, I'll try to change that, I already changed pretty much all the holder and the tube, but I didn't touch the washer yet. I hope that this will help me.

Thanks William,

I'm opening the holder every morning for chlorating the electrode, so I know how to assemble it. Anyway thanks a lot for your feedback, I'll also try with another pin cap!