Sep 15, 2022

Public workspaceProtocol for ex vivo patch clamping

DOI
dx.doi.org/10.17504/protocols.io.261ge361ol47/v1
  • Maia Datunashvili1
  • 1Northwestern university
divya.darwinarulseeli
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DOI: dx.doi.org/10.17504/protocols.io.261ge361ol47/v1
Protocol Citation: Maia Datunashvili 2022. Protocol for ex vivo patch clamping. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge361ol47/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: September 15, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70106
Keywords: ASAPCRN
Abstract
Protocol for ex vivo patch clamping
Materials
Reagents, instruments and materials
  • Ketamine / xylazine cocktail
  • Fine science surgical tools, including scalpel, scissors, forceps
  • Petri dishes
  • Vibratome
  • Blade

Solutions:
  • Sucrose-based artificial cerebrospinal fluid (sucrose aCSF in mM): 230 sucrose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 10 MgSO4, 10 glucose, 1 Na-pyruvate, and 0.005 L-glutathione.
  • Standard aCSF (in mM): 26 NaHCO3, 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 2 MgSO4, 10 glucose, 1 Na-pyruvate, and 0.005 L-glutathione.
  • Synthetic interstitial fluid (SIF) warmed to 35C, equilibrated with 95% O2 and 5% CO2 and containing (in mM): 26 NaHCO3, 126 NaCl, 3 KCl, 1.25 NaH2PO4, 1.6 CaCl2, 1.5 MgSO4, and 10 glucose.

Equipment Setup
  • Vibratome
Preparation of acute brain slices
Preparation of acute brain slices
Chill sucrose aCSF up to 4 degrees and then keep it on the ice and bubble continuously by carbogen.
Place 150 ml standard aCSF in holding chamber. Warm it up to 34oC in water bath and bubble continuously by carbogen.
After 3-5 weeks of the viral injection, anesthetize the animal briefly with Isoflurane and then make IP injection of ketamine/xylazine cocktail (87/13 mg/kg i.p.).
Perfuse the animal transcardially with 4-6 ml ice-cold sucrose aCSF.
Open the scalp, block the brain between the hemispheres and transfer it to the petri dish filled with ice-cold oxygenated sucrose solution.
Glue the brain on the specimen plate and cut 250 um thickness slices on the vibratome.
Wash the slices with standard aCSF and transfer them to the holding chamber. Keep the slices 30 min at 340C and then at room temperature.