Protocol for ex vivo patch clamping
Maia Datunashvili
Abstract
Protocol for ex vivo patch clamping
Steps
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.
