May 09, 2026
Ex Vivo Mouse Brain Slice Whole-Cell Patch-Clamp Electrophysiology
- Andrew Miller-Hansen1
- 1UCSF
Protocol Citation: Andrew Miller-Hansen 2026. Ex Vivo Mouse Brain Slice Whole-Cell Patch-Clamp Electrophysiology. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl84yq7g2w/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: May 08, 2026
Last Modified: May 09, 2026
Protocol Integer ID: 316604
Keywords: ASAPCRN, ex vivo mouse brain slice whole, clamp electrophysiology this protocol, clamp electrophysiology, optogenetic stimulation, clamp electrophysiology experiment, vivo slice, cell patch, stimulation, voltage clamp, including voltage clamp, mouse brain, viral injection, current clamp recording, clamp recording, vivo, channelrhodopsin2, such as channelrhodopsin2, encoded opsin, mice, transgenic expression, ex vivo mouse
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020529
Abstract
This protocol outlines steps for performing ex vivo slice whole-cell patch-clamp electrophysiology experiments. For optogenetic stimulation, this procedure is typically performed on mice expressing genetically encoded opsins, such as Channelrhodopsin2, which can be introduced through viral injection or transgenic expression.
Attachments
Materials
- ACSF components (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 15 glucose, 2 CaCl₂, and 1 MgCl₂.
- Cs-based internal solution components (in mM): 115 CsMeSO₃, 10 TEA, 10 HEPES, 5 QX-314 chloride, 4 Mg-ATP, 0.4 Na₃-GTP, 10 Na₂-phosphocreatine, 1 EGTA, 0.1 CaCl2.
- K-based internal solution components (in mM): 135 KMeSO3, 5 KCl, 10 HEPES, 4 Mg-ATP, 0.3 Na3-GTP, 8 Na2-phosphocreatine, 1 EGTA, 0.1 CaCl2
- 0.22 µm membrane filter (e.g. MilliporeSigma‱).
- Vibratome (e.g., Leica VT1200s)
- Patch-clamp recording set up (specific components listing in protocol for Ding lab)
- 450 nm light laser (e.g. Opto Engine LLC) and a 594 nm light (e.g. Opto Engine LLC)
Safety warnings
Wear appropriate PPE as required by your institution.
Ethics statement
Prior ethics approval (e.g. IACUC) should be obtained before performing these experiments. Approval was obtained by the Stanford University IACUC before any procedures were performed.
Prepare artificial cerebrospinal fluid (ACSF)
Prepare 1x ACSF and NMDG slicing solution fresh daily before slicing. Slicing colution contains (in mM): 92 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 Glucose, 5 Na-Ascorbate, 2 Thiourea, 3 Na-Pyruvate, 10 MgSO4, 0.5 CaCl2 (Millipore Sigma).
ACSF for recovery and recordings contained in mM: 125 NaCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, 11 Glucose. For recordings, picrotoxin (50 μM, Sigma) was added.
Continuously bubble the ACSF solution with 95% O₂ and 5% CO₂ to maintain pH at 7.4 and osmolarity at 300-305 mOsm.
Brain extraction and slicing
Anesthetize mice following protocols approved by the Animal Care and Use Committee. Transcardially perfuse with ice-cold N-Methyl-D-Glucamine (NMDG) solution.
Decapitate the animal, immediately extract the brain, and submerge it in ice-cold ACSF or other optimized cutting solutions (e.g. high sucrose, low-sodium solution).
Use a vibratome (e.g., Leica VT1200s) to prepare 300 µm-thick coronal slices containing the region of interest.
Prepare either a CS-based internal solution for whole-cell voltage clamp recordings
or K-Based internal solution for whole-cell current clamp recordings. This can be done ahead of time.
Cs-based internal solution 13 steps
Prepare either a CS-based internal solution for whole-cell voltage clamp recordings
or K-Based internal solution for whole-cell current clamp recordings.
Prepare cesium-based internal solution containing in mM: 117 CsMeSO3, 2.8 NaCl, 0.4 EGTA, 20 HEPES, 4 Mg-ATP, 0.3 Na3-GTP, 5 QX314 Cl, 5 TEA-Cl, 0.1 Spermine 4HCl.
Adjust the solution to pH 7.3-7.4 with CsOH and osmolarity at 280-290 mOsm.
After fully dissolving, filter the solution twice using a 0.22 µm membrane filter (e.g., MilliporeSigma‱).
Store in small aliquots (e.g.100 µL per tube) at -20°C.
Patch-clamp recording setup
Transfer a slice to a recording chamber, secure with an anchor (e.g., Warner Instrument), and begin ACSF perfusion at 2-3 mL/min at 30°C.
Mount the chamber on a microscope (e.g. Olympus BX51), equipped with a Multiclamp 700B amplifier (e.g., Molecular Devices) and monitored by custom MATLAB software (Mathworks) or other recording software (e.g. Wavesurfer).
Use glass pipettes (3-5 MΩ) prepared with a micropipette puller (Narishige, PC-100), filled with the appropriate internal solution (e.g., Cs-based).
Apply positive pressure (~5 Kp) to the glass pipette while approaching the targeted cell. Upon contact, remove pressure to form a “Giga Ohm seal.”
Once the seal is formed, hold voltage at -70 mV and apply brief negative pressure to achieve whole-cell patch mode (“Break-in”).
Low-pass filter the signal at 2.2 kHz and digitize at 10 kHz.
Exclude cells with series resistance >25 MΩ from analysis for quality control.
Recording optogenetically evoked EPSCs
Once a whole-cell patch is established, hold the membrane potential at -70 mV for excitatory postsynaptic currents (EPSCs) dependent on AMPARs and at +40mV for NMDAR/AMPAR currents.
To measure AMPA rectification indices, i.e. for separation of +40 AMPA from NMDA currents, wash in D-AP5 (50 μM, Tocris).