Jan 09, 2026
Whole-cell patch-clamp recordings
- Hongfei Xu1,
- Akio Mori1,
- Robert Edwards1
- 1University of California San Francisco
Protocol Citation: Hongfei Xu, Akio Mori, Robert Edwards 2026. Whole-cell patch-clamp recordings. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpmnxpgzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 20, 2025
Last Modified: January 12, 2026
Protocol Integer ID: 233013
Keywords: preparation of hippocampal slice, hippocampal slice, clamp recording, cell recording, ca1 pyramidal neuron, clamp recordings this protocol, neuron, cell patch, mediated epsc, cell, stimulation, stimulation in the stratum radiatum
Abstract
This protocol describes the preparation of hippocampal slices from mice for whole-cell patch-clamp recordings. Whole-cell recordings are made from CA1 pyramidal neurons, with AMPAR-mediated EPSCs evoked by stimulation in the stratum radiatum and monitored under voltage-clamp conditions.
Materials
BuffersCutting aCSF
- 210 mM sucrose
- 2.5 mM KCl
- 0.5 mM CaCl₂
- 7 mM MgSO₄
- 1.25 mM NaH₂PO₄
- 25 mM NaHCO₃
- 7 mM D-glucose
- 1.3 mM ascorbic acid
- Saturate with 95% O₂/5% CO₂
- pH 7.4, 305–310 mOsm
Recording aCSF
- 119 mM NaCl
- 2.5 mM KCl
- 2.5 mM CaCl₂
- 1.3 mM MgSO₄
- 1 mM NaH₂PO₄
- 26.2 mM NaHCO₃
- 11 mM d-glucose
- Saturate with 95% O₂/5% CO₂
- pH 7.4, 305–320 mOsm
Pipette solution
- 135 mM CsMeSO₃
- 8 mM NaCl
- 0.3 mM EGTA
- 4 mM Mg-ATP
- 0.3 mM Na-GTP
- 5 mM QX-314
- 0.1 mM spermine
- 10 mM HEPES
- pH 7.4, 290–295 mOsm
Troubleshooting
Patch pipette preparation
Pull pipettes from borosilicate glass using a Flaming/Brown puller (P-87).
Fill pipettes with pipette solution; typical resistance 3–5 MΩ.
Animal preparation and brain slicing
Anesthetize P21–P35 mice with ketamine/xylazine (i.p.).
Perfuse with ice-cold cutting aCSF saturated with 95% O₂/5% CO₂.
Dissect brains in ice-cold cutting aCSF. Slice into 300 μm near-horizontal sections using a vibratome in oxygenated cutting aCSF. Transfer slices to a holding chamber containing recording aCSF.
Incubate slices at 32°C for 30 min, then maintain at room temperature until recording. Perform a micro-cut between CA1 and CA3 to prevent network propagation.
Whole-cell recordings
Perform recordings in recording aCSF saturated with 95% O₂/5% CO₂ at 34 ± 0.5°C, perfusion rate 3 mL/min.
Evoke AMPAR-mediated responses at -70 mV holding potential in the presence of 10 μM AP-V and 100 μM picrotoxin.
Fill the recording pipette with intracellular solution and check the recording pipette under the microscope to remove any residual air bubbles.
Apply positive pressure using 1ml surge to clear the path, and once the recording electrode approaches the cell, the positive pressure will create a dimple as it touches.
Remove the positive pressure immediately and watch for a slight resistance increase as positive pressure relieve.
Apply gentle suction and monitor pipette resistance; once it exceeds 1 GΩ, hold the cells at -68 mV immediately and release the suction.
Apply stronger, brief negative suction to rupture the membrane patch.
Note: The pipette now has access to the cell's interior, forming the whole-cell configuration.
Allow intracellular solution to exchange (2 mins) before recording.
Stimulate the stratum radiatum using a tungsten bipolar electrode.
Monitor membrane current, input resistance, and series resistance. Discard cells if series resistance exceeds 30 MΩ. Accept recordings only if seal resistance >1 GΩ.
Data acquisition
Acquire data with MultiClamp 700B amplifier.
Filter at 2 kHz and digitize at 10 kHz. Store and analyze data using IgorPro v6.3.4.1. Peak EPSC amplitudes are determined from recorded currents