Morphological artifacts induced in intracellularly stained neurons by dehydration: circumvention using rapid dimethyl sulfoxide clearing.

In order to observe the fine details of intracellularly stained neurons in brain slices the slices must be cleared of opaque matter. This clearing process involves dehydration of the slice, which typically results in significant shrinkage of the cleared tissue. However, how this shrinkage affects neuronal morphology has not been demonstrated to date. In this paper we detail the artifacts induced in the morphology of stained neurons by this clearing process. During dehydration-induced shrinkage of the brain slices, neurons stained with the water-soluble dye, Lucifer yellow, demonstrated a dramatic decrease in size to less than two-thirds of their original dimensions. In contrast neurons stained with the horseradish peroxidase/diaminobenzidine reaction-product did not shrink with the slice; instead the dendrites bent and curled during dehydration with no loss in cell-soma size. We have managed to circumvent these artifacts by using as a clearing agent the solvent dimethyl sulfoxide, which is miscible in both aqueous and organic phases. This solvent will clear tissue slices without inducing the concomitant artifacts caused by tissue shrinkage occurring with the alcohol-dehydration process.