Acute Brain Slices

Set up vibratome and cutting station:

Make 1X ACSF:

• Prepare 1 L per mouse.
• 900mL + 100mL + 1.8g + 2g
• Keep in water bath 32°C and bubble continuously with 95% O₂ / 5% CO₂.
• Add ~30 mL to a slice holding vial, keep at 32°C and bubble continuously with 95% O₂/ 5% CO₂.

Make 1X cutting solution:

• Prepare 250 mL per mouse
• 225mL + 25mL + 0.5mg + 0.125g + 4.3g
• Chill on ice and bubble with 95% O₂ / 5% CO₂.

Set up vibratome

• Carefully clean blade with ethanol and water
• Using a hex wrench, insert blade in blade holder assembly
• Run VibroCheck calibration
• Place buffer tray in ice tray and fill ice tray with ice; load on to vibratome
• Superglue a rectangular piece (~2 cm x 1 cm x 1 cm) of 3% agar onto the specimen plate

Set up perfusion and cutting tools

Perfuse animal with cold cutting solution

Dissect brain and glue it to specimen plate.

Insert plate in buffer tray and fill tray with 1X cutting solution.

Lower blade into solution, set front and back limits and settings.

• Slice at 0.8 mm/s
• 240 µm slices

Collect relevant slices as they come off the blade, and carefully transfer to warmed slice vial.

Recover slices at 32°C and bubbled for at least 45 minutes.

10X Cutting solution stock

A	B	C	D	E
Compound:	CAS No.	FW:	conc. (mM)	10X Stock (g/2L)
NaCl	7647-14-5	58.44	75	87.66
KCl	7447-40-7	74.55	2.5	3.72
MgCl2*6H2O	7791-18-6	203.3	6	24.4
CaCl2*2H2O	10035-04-8	147.01	0.1	0.3
NaH2PO4*H2O	10049-21-5	137.99	1.2	3.32

10X ACSF stock

A	B	C	D	E
Compound:	CAS No.	FW:	conc. (mM)	10X Stock (g/4L)
NaCl	7647-14-5	58.44	126	294.52
KCl	7447-40-7	74.55	2.5	7.44
MgCl2*6H2O	7791-18-6	203.3	1.2	9.75
NaH2PO4*H2O	10049-21-5	137.99	1.2	6.64
CaCl2*2H2O	10035-04-8	147.01	2.5	14.7