I usually use glycerol-based ACSF for cutting brain slices from adult mice. Sometimes the preparation results very hard because when I add the last substance (NaHCO3) the solution precipitate (not always). Do you know why it happens? The second problem is about the osmolarity: using the following preparation, I usually obtain a solution of 330-340 mOsm that I have to dilute for obtaining an osmolarity of 300 mOsm. Is the procedure correct?
The solution contains (in mM): 250 glycerol, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 26 NaHCO3 and 11 glucose.
For the first half of the question, it depends on what you are doing with the slices, in the lab I work in we use thick mouse brain slices for electrochemistry so we keep them oxygenated. We don't use glycerol in the aCSF but this might be something you need for your preparation.
aCSF : NaCl 126mM, KCl 2mM, KH2PO4 1.4mM, MgSO4 2mM, NaHCO3 26mM, CaCL2 2.4mM, (+)Glucose 10mM.
As for the precipitation, it is the calcium that causes the problem, put the calcium in last after the everything else has totally dissolved.
Make sure your solutions are fresh. Add calcium and magnesium after you bubble your solution with carbogen gas, at least 10 minutes. I suspect bubbling is not going to work well with glycerol and without correct pH calcium and magnesium will precipitate. Another alternative may be to use of HEPES instead of NaHCO3 and KH2PO4, then you will only need to adjust your pH, before adding calcium and magnesium.
I use glycerol aCSF very often for cutting brain slice. Firstly, to avoid solution precipitating you'd better put CaCl2 and MgCl2 into the solution finally. Dissolving all other compounds in the solution then speed up the stirrer and slowly put CaCl2 and MgCl2. Secondly, the osmolarity of glycerol aCSF is about 330~340 mOsm. I just diluted a little bit and keep glycerol aCSF 325-330 mOsm. A little higher osmolarity is good for heart perfusion and cutting brain slice to prevent tissue from swelling.
Good Luck!
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