Are the capillaries with or without filament? You might want to try without filament. Based on my experience, you will have a better access. In addition, I usually combine neg. pressure with zap. Still strange that the access gets worse with a new filament. I guess that you did the necessary initial steps to evaluate the new filament (ramp test)? Best, Jakob

If you're able to get gigaohm seals and the electrodes look okay under 40x -- meaning both that the shape is okay and that there are no visible bubbles or debris in solution clogging the tip -- then I doubt that your new filament is the culprit. Instead, I would suggest checking your internal solution and ACSF.

In particular, I would check the osmolality and pH. When I do cortical slices, I typically aim for 290 mOsm internal and 310-320 mOsm ACSF. Both solutions have a pH of just over 7.4 at room temperature; the pH will go down when you warm them to physiological temperature.

You might also try using mouth suction to breakthrough rather than a syringe. Doing it by mouth tends to allow you to deliver a much sharper bolus of negative pressure than you can achieve by doing it manually with a syringe. When I've measured pressure with a manometer, I find that by mouth I tend to deliver -2 psi (or even more) in about half a second.

One possibility is releasing the neg. pressure, you can changing from .15 or .25 to 0 mL of neg pressure constantly (0.5 Hz) until you can reach a decent Ra, sometimes if you apply slight positive pressure (when you are trying to get back to 0 mL) it can help to get the patch broken.

One possibility I can think of is that you may be locating the tip of your pipette now at an inconvenient place on the cells. The best place is on top of the centre of the cell body. You may be able to get a gigaseal but not break in if you are off the centre. Another possibility is that the tubing for suction is faulty, and I would check that carefully.

I recommended Niraj's answer and Thor's. They gave you great tips. Osmolarity and checking your suctions tubing. I only will add to also check the pipette holder for any cracks (especially the Axon pip. holders are subject to cracking) and you can also change the washer.

If you still can't achieve WC config. then go back to your best model (maybe younger cells from younger animals) where you used to have easy breaks an test your system again with the new filamented glass.

If you polish your tips, don't make the tip too small as you will have more trouble breaking into the cell.

Hi Nikolaj, I hope you resolved your problem by this time.

If not, I think Dr Jakob von Engelhardt already gave you the answer. Every time you change the filament is necesary to run a Ramp test and adjust the protocol in base the new melting point (it may be very similar that the last you had). The issue in here is that a slight change in the filament's shape and position,will change the general shape of the pipette, even when the tip and pipette resistance might seem good or similar to the one you had before. In this case, you may have same -Rp short taper- in a thinner pipette, which require more sucction. When this happen, if you are not aware of it, ususaly won't have trouble with getting the Gseal but the W-C as it may cause to suck in a lot more membrane into the pipette.

PS: The better shaped and positioned the fillament, the lesser the troubles you get when resume patching.Therefore, is better to spend the necessary time in positioning the filament and adjusting the protocol.

Hope this help. Regards!.