Patch Clamp introductory visual tutorial

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--- Short overview of the Patch Clamp Technique using mice brain slices for our students -- • In this short movie we will walk through with Julia, freshly graduated master student, who will prepare slices and perform whole-cell recording. • made by Anna, PhD student //twitter: @AnTokarska// • Overview of the main steps - 00:28 • 1. Prepare all solutions in advance – ACSF and sucrose-based, protective ASCF for cutting and intracellular solution of your choice • 2. Slice preparation – 00:30 • a. Dissect the animal and remove the brain • b. Cut the slices using vibratome – in cold sucrose-ACSF - 00:43 • c. Transfer slices to slicing chamber and incubate for about 30min in 35stC - 01:03 • 3. Transfer slice to the recording chamber - 01:08 • 4. Prepare the set up and start perfusion system (warm ASCF for mice tissue) • 5. Pull micropipettes – 01:32 • 6. Fill pipette with internal solution, make sure there are no bubbles – 02:02 • 7. Place pipette in the holder so the solution has connection with silver chloride coated wire electrode inside - 02:09 • 8. Use the micromanipulator to change the focus of the microscope and visualize the slice and localize the cell of interest – 02:12 • 9. Overview of the equipment used to obtain the signal – 02:22 • 10. Place pipette under the objective and use the micromanipulator to locate the micropipette and focus on the tip (still above the slice) – 02:36 • 11. Measure the micropipette resistance by applying a voltage step – seen as rectangle step in pink trace on oscilloscope - 03:07 • 12. Apply positive pressure using syringe and slowly approach the cell – 03:15 • 13. Use negative pressure to obtain gigaseal and open the cell, observe change of the resistance visible on the oscilloscope – 03:20 • 14. Perform voltage or current-clamp recordings using the computer software – 03:28 • 15. If you have any light-sensitive opsins you can check for fluorescence or stimulate them with LED/lasers – 03:42

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