When we were first getting started with the CLARITY technique, we perfused a lot of brains at once and left them in the hydrogel at 4 degrees until we were ready to embed them. When we switched to brains that had been injected with GFP-labeled PRV and to Thy1-EGFP brains, we did the same thing. What we've been seeing is very good structural preservation, a load of autofluorescence, and no GFP signal at all. We're assuming that overfixation (beyond the 2 days post-fix in hydrogel as stated in the published protocol) is the cause of our signal loss. Has anyone else experienced this?