Tissue must be prepared as follows:
1. Mice were anesthetized with a lethal dose of avertin (1.25% solution in water or 12.5mg/mL in water).
2. After mice are no longer responsive to toe pinch, they were transcardially perfused with 1X Tris Buffer Saline (TBS) until they run clear (~ 5 minutes).
3. Following transcardial perfusion with 1X TBS, mice were then perfused with 4% Paraformaldehyde (PFA) dissolved in 1X TBS for ~ 5 minutes (until stiff).
4. Finally, brains were dissected from the mouse and stored overnight (~12-18 hours) at 4 degrees Celsius in 4% PFA (enough to cover the brain).
5. The following day, brains were removed from PFA, rinsed with 1X TBS, and then stored at 4 degress Celsius in a 30% sucrose solution in 1X TBS.
6. The brains are stored until they sink to the bottom of their container and can then be frozen and cryosectioned.
Any alterations to tissue preparation should be tested empirically to be certain the resulting staining is still comparable to the staining resulting from the previously described tissue preparation.
In brief, the final solution is 50mM N-Propyl Gallate, 20 mM Tris, and 90% Glycerol in water.