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

Protocollo

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.

Materiali

Name	Company	Catalog Number	Comments
Compresstome VF-200	Precisionary Instruments	VF-200	Vibrating tissue slicer (recommended)
N-methyl-D-glucamine	Sigma Aldrich	M2004	aCSF constituent
Sodium Chloride	Sigma Aldrich	S3014	aCSF constituent
Potassium Chloride	Sigma Aldrich	P5405	aCSF constituent
Sodium Phosphate monobasic dihydrate	Sigma Aldrich	71505	aCSF constituent
Sodium Bicarbonate	Sigma Aldrich	S5761	aCSF constituent
HEPES	Sigma Aldrich	H4034	aCSF constituent
Glucose	Sigma Aldrich	G7021	aCSF constituent
Sodium Ascorbate	Sigma Aldrich	A4034	aCSF constituent
Thiourea	Sigma Aldrich	T8656	aCSF constituent
Sodium pyruvate	Sigma Aldrich	P5280	aCSF constituent
Calcium chloride dihydrate	Sigma Aldrich	C7902	aCSF constituent
Magnesium Sulfate heptahydrate	Sigma Aldrich	M1880	aCSF constituent
Curved blunt forceps	Fine Science Tools	11065-07	Brain dissection tools
Fine dissecting scissors (supercut)	Fine Science Tools	14058-09	Brain dissection tools
Large heavy duty scissors 7''	Fine Science Tools	14000-18	Brain dissection tools
Metal spatula	Sigma Aldrich	Z511455-1PAK	Brain dissection tools
Razor blades	VWR	89031-954	Brain dissection tools
Brain Slice Keeper-4	Automate Scientific	S-BSK4	brain slice holding chamber
nylon netting	Warner Instruments	64-0198	For building small slice recovery chambers
Pyrex glass beakers (250 mL)	VWR	89090-434	For building small slice recovery chambers
35 mm plastic dish, round	VWR	100488-376	For building small slice recovery chambers
Gas diffuser stones (10 µm)	Sigma Aldrich	59277	For constant carbogenation (fine bubbles)
Agarose Type I-B	Sigma Aldrich	A0576	For embedding brain specimens
Hydrochloric acid	Sigma Aldrich	H1758-100ML	For pH adjustment of media
Sodium Hydroxide	Sigma Aldrich	221465-25G	For pH adjustment of media
Potassium Hydroxide	Sigma Aldrich	221473	For pH adjustment of media
Plastic transfer pipets 3 mL graduated	VWR	89497-676	For slice transfer
Zirconium ceramic injector blades	Cadence Specialty Blades	EF-INZ10	http://cadenceinc.com/
Heated water bath (2.5L)	VWR	13491-060	Miscellaneous
Filter paper rounds	VWR	28456-022	Miscellaneous
Cyanoacrylate glue	Amazon	B000BQRBO6	Miscellaneous
Glass petri dish	VWR	89000-326	Miscellaneous
10X Phosphate buffered saline	Sigma Aldrich	P5493	Miscellaneous
Thermomixer (w/1.5 mL tube block)	VWR	89232-908	To keep agarose molten