Ex vivo Brain Slice Preparation for electrophysiology and 2PLSM recordings

On the experiment day prepare the working aCSF and slicing solution in a 1:10 dilution from stock solutions

Place slicing solution in -20°C freezer for 30-40 minutes.

Then place the slicing solution in a large Styrofoam container of ice to remain cold.

Place two 95mm diameter glass petri dishes on top of ice bucket and fill with cold slicing solution. Bubble the slicing solution in the bottle and the two petri dishes.

Turn on water bath and set to 34°C°.

Fill a 250 mL beaker with 150mL of slicing solution.

Place a second container that has a sieve bottom into the beaker containing the slicing solution.

Place the beaker with the slicing solution and second container in water bath and begin bubbling it.

Fill a 95mm diameter glass petri dish with slicing solution and set aside.

Cut a 1 cm piece of the 20° agarose wedge and super glue it to the specimen plate.

Insert razor blade into blade holder.

Set parameters (speed, amplitude, and section thickness) on the Vibratome control panel.

Place ice in the chamber below the specimen plate and prepare a glass with water next to the vibrotome.

Fill one 10 mL syringe with cold slicing solution, connect with tubing, and place 27G needle at end of tubing.

Fill a styrofoam tray with ice.

Anesthetize the mice with a mixture of ketamine (50 mg/kg) and xylazine (4.5mg/kg).

Use toe pinch-response method to determine depth of anesthesia.

Place the animals on ice in tray lying on the back with face upward.

Make an incision through the abdominal skin.

Make two additional skin incisions from the xiphoid process along the base of the ventral ribcage laterally.

Gently reflect the two flaps of skin to expose thoracic field completely.

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.

Separate the diaphragm from the chest wall on both sides with scissor cuts.

Clamp the reflected ribcage laterally with a hemostat to expose the heart.

Secure the beating heart with fingers or blunt forceps, and immediately insert a blunt 27G syringe needle.

Cut the right atrium with scissors, and at the first sign of blood flow, begin the infusion of 1x slicing solution at 2-4 ml/min.

Continue perfusion with slicing solution until the 10 mL syringe is empty.

Decapitate the mouse with large surgical scissors.

Place the decapitated head in one of the petri dishes containing cold, bubbling slicing solution. Removal of brain must be done in cold, bubbling slicing solution.

Cut down the midline to expose the skull.

Make two lateral and one dorsal cut using sharp surgical scissors on the base of the skull.

Cut the olfactory bulbs/optic nerve at rostral end of skull.

Gently peel off the skull using blunt forceps.

Once brain is fully exposed, remove it from the skull and place it in a petri dish with clean, cold and bubbled slicing solution.

Cut the brain in two halves along the midline.

Super glue the flat section (midline) onto the agarose wedge glued to the specimen plate so that the vibratome cuts along parasagittal plane with a 20° angle. Place the posterior part of the brain in the top of the agarose wedge. If bilateral sections are needed, super glue both hemisferes to the agarose wedge.

Place specimen plate in buffer tray and fill buffer tray with slicing solution.

Place O2/CO2 tubing into buffer tray to bubble slicing solution.

Play start to the vibrotome and cut the desired slices.

With the glass transfer pipette, transfer the slices to the beaker containing the sieve bottom container at 34°C.

When finishing slicing, turn of the water bath and let it cool to room temperature.

Leave brain slices in beaker for 1h 0m 0s to recover.