SeeDB was described by Ke et al., 2013. Nat. Neurosci. 16, 1154-1161 [doi:10.1038/nn.3447]. SeeDB is saturated (~80% w/w) fructose in water supplemented with 0.5% (w/w) 1-thioglycerol (α-thioglycerol) to suppress the Maillard reaction, which would lead to the browning of the specimen and enhanced autofluorescence.

In Helobdella embryo, SeeDB is the only clearing reagent that has been tested to preserve phalloidin labeling over an extended period of time.

SeeDB is highly viscous and a lot more difficult to handle than other aqueous clearing solutions, but SeeDB appears to work better than Scale and buffered glycerol in terms of its clearing effect on the yolky early Helobdella embryo. We make only a small amount of SeeDB solution each time. Mix 5 ml of ddH₂O (or PBS, to reduce the expansion of specimen) and 100 μl of 1-thioglycerol. Then, add 20 g fructose gradually into the solution. Mix by shaking in 37°C until the solution becomes clear and viscous. Store the solution at room temperature.

From the stock, make ~1/2, ~1/4 and ~1/8 serial dilutions of SeeDB in PBS.

Transfer the specimen gradually from 1/8 SeeDB to 1/4 SeeDB to 1/2 SeeDB and finally to full-strength SeeDB. Incubate for 1-2 hour in for each step.

Keep the specimen in See DB overnight before mounted for observation. Note that due to high density and viscosity of SeeDB, care must be taken for handling and mounting specimen in SeeDB. The leech embryo would float in the SeeDB syrup, rather than settling down on the bottom. Thus, excessive SeeDB must be avoided when mounting specimen under a coverslip, otherwise, your valuable specimen will be crushed or pushed out of the coverage of the coverslip.