TITLE: Brain Slice Preparation for electrophysiology recording
AUTHORS: Hong-Yuan Chu, Hong-yuan Chu

[DESCRIPTION]
This protocol describes brain slice preparation for electrophysiology recording.

[BEFORE_START]
Before beginning

Wear PPEs before entering patch clamp room when making brain slice. i.e. gloves, coat, and face mask. 
Make sure the sharps container is not full. Replace if full.

[STEPS]
SECTION: Prepare Bubbling Solutions
1. On the prior day, prepare the working aCSF and sucrose aCSF solution in a 1:10 dilution from stock solutions (1 part stock, 1 part bicarbonate, 8 part Milli-Q water). 
 

SECTION: Prepare Bubbling Solutions
2. On day of cutting, place sucrose aCSF solution in -20 °C freezer for 30-40 minutes.
SECTION: Prepare Bubbling Solutions
3. Remove sucrose aCSF and place in large Styrofoam container of ice to remain cold.
SECTION: Prepare Bubbling Solutions
4. Place two 95mm diameter glass petri dishes on top of ice bucket and fill with cold sucrose aCSF. Bubble the sucrose aCSF in the bottle and the two petri dishes.
SECTION: Prepare Bubbling Solutions
5. Turn on water bath and set to 35 °C.

SECTION: Prepare Bubbling Solutions
6. Fill a 250 mL beaker with 150 mL of working aCSF.

SECTION: Prepare Bubbling Solutions
7. Place a second container that has a sieve bottom into the beaker containing the working aCSF.
SECTION: Prepare Bubbling Solutions
8. Place the beaker with the aCSF and second container in water bath and begin bubbling working aCSF.
SECTION: Prepare Bubbling Solutions
9. Fill a 95 mm diameter glass petri dish with working aCSF and set aside.
SECTION: Prepare Vibratome
10. On the cutting head of the vibratome, rotate blade holder 45°-90° using a size 3 Allen key.
SECTION: Prepare Vibratome
11. Open the blade holder using a size 3 Allen key and insert razor blade into blade holder.
SECTION: Prepare Vibratome
12. Clamp down the blade holder using the size 3 Allen key until hand-tight.
SECTION: Prepare Vibratome
13. Set parameters (speed, amplitude, and section thickness) on the Vibratome control panel.
SECTION: Prepare Vibratome
14. Turn on Julabo FL300 (recirculating chiller/cooler) and set to desired temperature to cool buffer tray.
SECTION: Prepare Vibratome
15. Cut a small piece of agarose (approximately 2x2cm square) and super glue to specimen plate so that it is standing on its side.
SECTION: Preparing Apparatus and Anesthesia
16. Fill one 10 mL syringe with cold sucrose aCSF, connect with tubing, and place 27G needle at end of tubing.
SECTION: Preparing Apparatus and Anesthesia
17. Fill metal tray with ice.
SECTION: Preparing Apparatus and Anesthesia
18. Weight the mouse to the nearest 0.1 gram. Anesthetize with Avertin (dosage: 0.3 mL/10 g body weight, i.p.). Place the mouse back to the home cage.

SECTION: Preparing Apparatus and Anesthesia
19. Use toe pinch-response method to determine depth of anesthesia.
SECTION: Preparing Apparatus and Anesthesia
20. Place the animals on ice in metal tray lying on the back with face upward.

SECTION: Perfusion Surgery
21. Make an incision through the abdominal skin.
SECTION: Perfusion Surgery
22. Make two additional skin incisions from the xiphoid process along the base of the ventral ribcage laterally.
SECTION: Perfusion Surgery
23. Gently reflect the two flaps of skin to expose thoracic field completely.
SECTION: Perfusion Surgery
24. Grasp the cartilage of the xiphoid process with blunt forceps and raise it slightly to insert pointed scissors. Cut through the thoracic musculature and ribcage between the breastbone and medial rib insertion points and extend the incision rostrally to the level of the clavicles.
SECTION: Perfusion Surgery
25. Separate the diaphragm from the chest wall on both sides with scissor cuts.
SECTION: Perfusion Surgery
26. Clamp the reflected ribcage laterally with a hemostat to expose the heart.
SECTION: Perfusion Surgery
27. Secure the beating heart with fingers or blunt forceps, and immediately insert a blunt 27G syringe needle. 
 

SECTION: Perfusion Surgery
28. Cut the right atrium with scissors, and at the first sign of blood flow, begin the infusion of 1x sucrose aCSF at 2-4 mL/min.

SECTION: Perfusion Surgery
29. Continue perfusion with sucrose aCSF until the 10 mL syringe is empty.
SECTION: Dissection
30. Decapitate the mouse with large surgical scissors.
SECTION: Dissection
31. Place the decapitated head in one of the petri dishes containing cold, bubbling sucrose aCSF. 
 

SECTION: Dissection
32. Cut down the midline to expose the skull.
SECTION: Dissection
33. Make two lateral and one dorsal cut using sharp surgical scissors on the base of the skull.
SECTION: Dissection
34. Cut the olfactory bulbs/optic nerve at rostral end of skull.
SECTION: Dissection
35. Gently peel off the skull using blunt forceps.
SECTION: Dissection
36. Once brain is fully exposed, make an incision along the coronal plane of the cerebellum to create a flat surface.
SECTION: Dissection
37. Super glue the flat section onto the specimen plate so that the vibratome cuts along coronal plane.
SECTION: Slicing
38. Place specimen plate in buffer tray and fill buffer tray with sucrose aCSF.
SECTION: Slicing
39. Place O2/CO2 tubing into buffer tray to bubble sucrose aCSF.
SECTION: Slicing
40. If using manual mode:
SECTION: Slicing
41. If using continuous mode:
SECTION: Slicing
42. Steps about continuous slicing.
SECTION: Slicing
43. After each slice, remove brain slice from buffer tray using a glass wide bore transfer pipette and place into the second petri dish on ice containing bubbling sucrose aCSF.

SECTION: Slicing
44. Once all brain slices are collected, move brain slices from the petri dish containing sucrose aCSF to a Room temperature petri dish containing working aCSF.

SECTION: Slicing
45. Move brain slices from the petri dish containing working aCSF to beaker in the water bath with bubbling working aCSF.
SECTION: Slicing
46. After 30 minutes, turn off water bath and leave brain slices in beaker for 60 min to recover.

SECTION: Slicing
40.1. Press the “AUTO/MAN” button so that the bottom LED light is on, indicating semiautomatic/manual mode. In semiautomatic mode, only continuous slicing (SINGLE) is possible.
SECTION: Slicing
40.2. Position razor blade (using the blade forward/backward and “UP” / ”DOWN” buttons on control panel) at desired location and perform first cut by pressing the “RUN/STOP” button. 
 

SECTION: Slicing
40.3. Press “RUN/STOP” on the control panel after blade has successfully cut through the entire brain.
SECTION: Slicing
40.4. Return the blade to the initial position using the blade backwards button on control panel.
SECTION: Slicing
40.5. Turn Dial 2 (location next to “Section Thickness” button on control panel) to desired slice thickness.
SECTION: Slicing
40.6. Press “RUN/STOP” to perform second cut.
SECTION: Slicing
40.7. Press “RUN/STOP” again after blade has successfully cut through the entire brain.
SECTION: Slicing
40.8. Repeat steps 40.3-40.6 until desired about of tissue or brain region(s) is cut.
SECTION: Slicing
41.1. Press the “AUTO/MAN” button so that the top LED light is on, indicating automatic mode.
SECTION: Slicing
41.2. Press the “SINGLE/CONT” button to toggle between single stroke and continuous stroke.
SECTION: Slicing
41.3. Position razor blade (using the blade forward/backward and “UP”/”DOWN” buttons on the control panel) above the brain at the initial window edge.
SECTION: Slicing
41.4. Press the first cutting window button to set the first window edge.
SECTION: Slicing
41.5. Move the blade forward to the second window edge and press the second window cutting edge button.