Prepare 10X stock solution.

Composition of 1L of 10X Holding ACSF (in mM): 1150 NaCl, 25 KCl, 12.3 NaH₂PO₄, 100 d-glucose, 20 CaCl₂ and 20 MgSO₄.

Composition of 1L of 10X Holding ACSF (in g): 67.2 NaCl, 1.86 KCl, 1.47 NaH₂PO₄, 18.02 d-glucose, 2.94 CaCl₂ and 4.93 MgSO₄.

Dissolve all salts in 1L of ddH₂O to make 10X stock solution of Holding ACSF.

Dilute 10X solution to 1X for use in experiments.

Dilute 100mL of 10X solution to ~ 800mL with ddH₂O and add (in mM): 26 NaHCO₃, 2 thiourea, 5 Na-ascorbate and 3 Na-pyruvate; (in g): 2.18 NaHCO₃, 0.15 thiourea, 0.98 Na-ascorbate and 0.33 Na-pyruvate.

Add ddH₂O to final volume of 1L.

Bubble the solution with carbogen (95% O₂5% CO₂) for ~10 minutes.

After bubbling, measure and adjust pH of solution to ~ 7.3 with 12 N HCl (<1 ml).

Refrigerate until use.