Application of SeeDB Clearing Method to Xenopus laevis Tadpoles
There are several clearing techniques that have been developed over the past decade that differ in their method of action but the common aim of all methods is to reduce light scatter and increase transparency of tissue samples. Light scatter occurs when there are differences between the refractive indices of the lipid membranes and aqueous intracellular and extracellular fluids. All techniques seek to homogenize the refractive indices of the sample by replacing the aqueous intra- and extracellular fluids with a solution with a refractive index closer to that of the lipid membrane. SeeDB utilizes fructose, which when dissolved in water has a refractive index of 1.490 at 25° C, while the refractive index of lipid membranes is estimated to be between 1.42 and 1.45. This method has previously been applied to brain tissue samples from mice, rats, and songbirds but not to Xenopus brains. For this experiment, brains were dissected from 3 to 4 day old tadpoles and underwent the SeeDB protocol. After the three days of the protocol, the brains were visualized under both a dissecting and confocal microscope. Initial comparisons of SeeDB brains to untreated brains show an increase in transparency of SeeDB brains. The goal of developing a protocol for clearing Xenopus brains is to allow for the 3-D imaging of structures like dopamine clusters that are currently imaged in 2-D. The initial results of clearing trials are promising and the next step is testing the efficacy of the protocol when combined with immunostaining. One of the advantages of using fructose rather than high index solvents is that it does not extinguish fluorescent proteins which bodes well for its use in combination with immunostaining.
