Good evening/good morning everyone!

Right now, in my lab we are refurbishing a whole cell patch clamp system, unfortunately my electronics skills are quite poor and this is causing me some problems.

The amplifier is an Axopatch 200B, the headstage is an Axon CV203BU, and the acquisition board is a National Instruments BNC-2120.

The main problem concerns the reference electrode, which needs to be rebuilt since the one used previously is no longer available.

I built the first cable by tin soldering to a normal copper wire the pin for the headstage input to a silver wire chlorinated in bleach and coiled, the solder was coated with enamel to insulate it of the bath.

At first this type of wire worked, but at some point the device began to register -140 mV in the bath, with the pipette offset set to 5. I then tried to construct another wire in the same way, but it did not help.

Following directions from a different source than those on the first type of cable, I tried to construct another type by tin soldering a chlorinated silver wire and pin to a coaxial cable; I tried both twisting the cable into a spiral and leaving it straight and inserting it into a salt bridge (small plastic tube filled with 3% Agarose in 3M KCl). With the salt bridge, it goes from -140 mV to about -90 mV.

I tried removing the solder, changing silver wire and connecting it to the coaxial cable only mechanically (I had read that the silver-tin interface could be problematic), nothing changed. I also tried connecting, in another cable, both silver wire and solderless pin, but nothing changed. In the coaxial cable, I followed all precautions to connect only the center portion and not the shielding.

If I attach the cellular model to the headstage this marks a potential of about 0 mV under the same conditions, which should indicate that from the headstage to the amplifier there should be no problem. So the problem should be either in the holder or in the reference electrode, I focused on the reference electrode because the holder is not homemade and anyway changing the wire inside, disassembling it, cleaning it and trying another holder we had did not change anything.