Apr 15, 2026

Brain slice preparation for electrophysiology recording

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4ww52vo5/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815;
  • 3VIB-KU Leuven Center for Brain & Disease Research
Sarah van Veen
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.3byl4ww52vo5/v1
Protocol Citation: Sarah van Veen, Keimpe Wierda 2026. Brain slice preparation for electrophysiology recording. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4ww52vo5/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: December 13, 2024
Last Modified: April 29, 2026
Protocol  Integer ID: 115441
Keywords: ASAPCRN, brain slice preparation for electrophysiology, brain slice preparation, acute brain slice, clamp electrophysiology, electrophysiology, cell patch, brain
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol details the preparation of acute brain slices from 8-week-old mice for whole-cell patch-clamp electrophysiology. 
Materials
Dissection/perfusion and patching tools: 
  • Sedation box
  • Isoflurane
  • 10mL serological pipette
  • Ice bucket with ice  
  • Barbed forceps 
  • Large surgical scissors 
  • Small scissors 
  • Scalpel 
  • (compartmentalized) holding grids for slice recovery
  • Beakers for recovery and holding solution (250 ml)
  • Vibratome
  • Heating bath set at 32°C
  • Borosilicate glass tubes
  • Super glue 
  • Specimen plate
  • Harp (slice anchor) 
  • Electrophysiology setup equipped for whole cell patch clamp recordings
Preparation of solutions
Electrophysiology solutions are prepared as either 1x (choline chloride) or 10x (recording & holding acsf) stock solutions and (if required) diluted for use on the day of the experiment. NaHCO3 & D-glucose are added fresh during dilutions to working solutions. We use ~1 week of shelve life for all solutions at 4°C. 
Prepare 500 mL 1 x Choline Chloride solution (slicing and recovery solution).
ABCD
Choline chloride (cutting & recovery solution) 
  mM MW 1x, g/500 mL 
KCl 2.5 74.55 0.093
NaH2PO4.H2O 1.25 137.99 0.086
CaCl2.2H2O 0.5 147.02 0.037
MgCl2.6H2O 7 203.300.712
D-Glucose 10 180.16 0.901
Na-ascorbate11.6198.111.149
Na-pyruvate3.1110.040.171
NaHCO32684.011.092
Choline Chloride110139.627.679

Prepare 250 ml 1 x Holding solution (dilute and add NaHCO3 and D-Glucose)
ABCD
Holding solution
  mM MW 10x, g/L 
NaCl 126 58.44 73.6344 
KCl 3 74.55 2.2365
NaH2PO4.H2O 1 137.99 1.3799
CaCl2.2H2O 1147.02 1.4701
MgSO4.7H2O 6 246.48 14.7840
   Added to 1x, g/L
NaHCO32684.012.1843
D-Glucose 10 180.16 1.8016
Prepare 250 ml 1 x Recording solution (dilute and add NaHCO3 and D-Glucose)
ABCD
Recording solution
  mM MW 10x, g/L 
NaCl 127 58.44 74.2188
KCl 2.5 74.55 1.8638
NaH2PO4.H2O 1.25 137.99 1.7249
CaCl2.2H2O 2147.02 2.9402
MgCl2.6H2O 1 203.3 2.0330
   Added to 1x, g/L
NaHCO32584.012.1003
D-Glucose 25 180.16 4.5040
Prepare bubbling solutions
15m
On day of cutting, prepare holding and recordings acsf from 10x stock.
Place 250 mL of the choline chloride cutting solution in -20°C or -80°C freezer for 30-40 min or 10-15 min, respectively. Medium should be partly frozen when used.

Keep precooled cutting solution on ice and bubble with carbogen (at least 00:15:00 before use).

15m
Place beaker with other 250 mL choline chloride in 32 °C waterbath and bubble with carbogen.

Place beaker with 250 mL holding solution at Room temperature and bubble with carbogen.

Prepare vibratome
Insert cutting blade in vibratome, if feasible/available use blade alignment (e.g. vibrocheck).
Set parameters (speed (0.08-0.10 mm/s), amplitude (1.15-1.30 mm), and section thickness (250 µm) on the vibratome control panel. 
Add precooled cutting solution to dissection buffer tray (which is cooled using chiller or ice) and keep bubbling with carbogen.
Check availability/preparation before starting dissection
Dissection
Add 1-2 ml isoflurane onto tissue on bottom of sedation box and cover with lid.
After 2-3 minutes place animal in sedation box and wait until animal is stationary and is breathing slowly (after ~20-30 seconds)
Take the animal from the sedation box and immediately decapitate with large scissors.
Place the decapitated head on cuttingboard. Removal of brain must be done as quickly as possible, ideally within 30-45 seconds. 
Cut the skin down the midline to expose the skull.
Make an incision down the midline using sharp surgical scissors up to the front of the brain (up to olfactory bulb).
Make to small incisions perpendicular to the midline above the olfactory bulb.
Gently peel off the skull using blunt forceps, starting on top of the cerebellum.  Try to push/peel the skull away from the head.
Once brain is fully exposed, make an incision along the coronal plane in between the olfactory bulb and the rostral part of the cortex. 
Gently flip out the brain of the skull using a small spoon and drop it directly into the cold cutting solution in the vibratome.
Let the brain cool for 2-3 minutes in the cutting solution.
Dry a precooled specimen plate.
Take the brain from the cutting solution and place on a cutting board.
Remove access cutting solution and remove cerebellum.
Make sure the specimen disc is dry and put a drop and smear out a drop of superglue.
Place the brain on the superglue area with the rostral part up.
Add some cold cutting solution to the brain to cool and solidify the glue.
Slicing
Place specimen disc in buffer tray and position the cortex towards the blade.
Position razor blade at desired location/height and perform first cut by pressing the “RUN/STOP” button.  
After blade has successfully cut through the entire brain, remove rostral cut part.

Return the blade to the initial position using the blade backwards button on control panel. 
Reposition blade (500-800 um) and perform second cut.  
Repeat desired region of the brain (e.g. primary visual cortex) has been reached. 
Start cutting sections of 250 um of the required area.
After slice has been collected immediately place in recovery solution at 32°C.
Keep each slice in recovery solution for 6 minutes and then store them in the holding solution at room temperature. Make sure not to mix too much choline chloride solution into the holding solution when transferring the slices.
 After all slices have been collected and recovered, turn off water bath and leave brain slices in beaker with holding solution for 1 hour to fully recover. 
Recording
Wash acsf through the setup for 5 minutes and discard this volume.
For these experiments we added TTX (1 uM) and AP-5 (100 uM) to external recording solution to specifically focus on mEPSCs (recorded at -70 mV) and mIPSCS (recorded at 0 mV) in L2/3 of primary visual cortex. However, the above mentioned slice preparation method can be used for many different types of electrophysiological experimental designs tailored to the requirements of the ongoing project.
Recordings are done at 32-33°C
For recordings we used borosilicate glass recording pipettes (resistance 3.5–5 MΩ, Sutter P-1000).
Internal medium used to record mEPSCs and mIPSCs in primary visual cortex:
Choline chloride (cutting & recovery solution) 
  mM MW 1´, g/10 mL 
CsMSF126 228.00.288
Hepes10238.300.0238
MgCl2*6H2O2.5 203.310.00508
ATP disodium salt4 551.150.0220
GTP sodium salt0.4 523.180.00209
Creatine Phosphate10255.080.0255
EGTA0.6380.350.00228
QX-314 chloride5298.850.0149
Biocytin3372.480.03
Adjust pH to 7.25 with CsOH (1M)