Preparation of Brain Sections Using a NMDG-Based Protective Recovery Method

1. Brain Dissection and Slicing

1. Decapitate the animal. Use a scalpel to open the skin on the head and expose the skull cap.
2. Use fine super-cut scissors to cut away the skin over the skull cap and make small incisions laterally on either side at the caudal/ventral base of the skull. Make additional shallow cuts starting at the caudal/dorsal aspect of the skull, moving in the rostral direction up the dorsal midline taking care not to damage the underlying brain. Make a final 'T' cut perpendicular to the midline at the level of the olfactory bulbs.
   NOTE: Care must be taken to ensure no damage is done to the brain region(s) of interest. In particular, at no time should there be any compressive force applied to the brain itself.
3. Use the round-tip forceps to grasp the skull, starting at the rostral-medial aspect and peel back towards the caudal-lateral direction. Repeat for both sides to crack open and remove the dorsal halves of the skull cap to expose the brain. Gently scoop out the intact brain into the beaker of pre-chilled NMDG-HEPES aCSF. Allow the brain to uniformly cool for ~1 min.
4. Use the large spatula to lift the brain out of the beaker and onto the Petri dish covered with filter paper. Trim and block the brain according to the preferred angle of slicing and the desired brain region of interest. Work quickly to avoid prolonged oxygen deprivation during handling.
   NOTE: Many slicing angles are possible. The exact blocking method and slicing angle will depend on the exact brain region, cell type, and circuit to be studied.
5. Affix the brain block to the specimen holder using adhesive glue. Retract the inner piece of the specimen holder enough to withdraw the brain block fully inside. Pour the molten agarose directly into the holder until the brain block is fully covered in agarose. Clamp the pre-cooled accessory chilling block around the specimen holder for ~10 s until the agarose has solidified.
6. Insert the specimen holder into the receptacle on the slicer machine and verify proper alignment. Fill the reservoir with remaining pre-chilled, oxygenated NMDG-HEPES aCSF from the 250 mL beaker and move a bubble stone into the reservoir for the duration of slicing to ensure adequate oxygenation.
7. Adjust the micrometer to begin advancing the agarose-embedded brain specimen. Start the slicer and empirically adjust the advance speed and oscillation frequency to the desired level.
   NOTE: Both settings should be in the low range. For best results, a single pass of the blade arm should take approximately 20 s and the oscillation should produce a very smooth and gentle humming noise with no overt buzzing.
8. Continue advancing and slicing the tissue in 300 µm increments (or other preferred thickness) until the brain region of interest is fully sectioned; the total time for the slicing procedure should be less than 15 min.

2. Optimized NMDG Protective Recovery Procedure

1. Initial NMDG recovery step (critical step): Upon completion of the sectioning procedure, collect up all of the slices using a cut-off plastic Pasteur pipette and transfer them into a pre-warmed (34 °C) initial recovery chamber filled with 150 mL of NMDG-HEPES aCSF. Transfer all slices in short succession and start a timer as soon as all slices are moved into the recovery chamber.