We finally got it to work with viral injection.


This is a Thy1EYFP mouse cerebellum in which GCaMP6f (EGFP) was injected into the simplex/crus1 area via adeno-associated virus (AAV). The large cells are Purkinje cells (GFP). The paler cells in the center are mossy fibers (EYFP).

The brain was fixed in hydrogel overnight, then passively cleared in 50 ml of clearing solution in a 37 degree water bath, with almost daily solution changes, for 16 days. All but the white matter had cleared at this point. We used FocusClear (about 5 ml for the right hemisphere; the solution had been used briefly twice before). The hemisphere was in FocusClear for about 20 hours before imaging.

We used a 20x Zeiss lens (nothing special) on a 2-photon microscope and were able to resolve structures down to 1 mm.
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