Whole-cell Patch-clamp Recordings in Brain Slices

Transfer a brain slice from the recovery chamber using a plastic-trimmed pipette and place it on the coverslip in the recording chamber. Using the 4x objective, position the desired area in the center of the chamber and secure it with a slice hold-down (harp).

Switch to the 40x objective and lower the lens into the ACSF, then use fine adjustment to bring the tissue into focus. Locate and mark a target cell on the computer screen to guide the recording micropipette.

Using a 1 mL syringe and non-metallic micro needle with a dedicated filter, fill the micropipette with prepared internal solution while ensuring no air bubbles are present. Place the micropipette in an electrode holder so the solution contacts the silver chloride-coated wire electrode.

Apply positive pressure to the micropipette before immersing it in ACSF to prevent debris entry. Using the micromanipulator, guide the pipette toward the center of the chamber and locate it on the computer screen, measuring its resistance and clearing any bubbles with voltage steps.

Gradually lower the micropipette while focusing down, slowing to medium-low speed as it contacts the slice surface. Apply light positive pressure to clear debris, then approach the target cell until a dimple appears on the cell surface, and apply weak suction to form a gigaseal.

After gigaseal formation, compensate for fast and slow capacitance using the amplifier. Once the seal is stable above 1 GigaOhm, apply a brief and strong suction pulse to rupture the plasma membrane and establish the whole-cell configuration.

Switch to cell mode and measure key parameters including input resistance, series resistance, and membrane capacitance. Continue monitoring these parameters throughout the recording session to assess cell health and signal quality.