Optimisation and validation of hydrogel-based brain tissue clearing shows uniform expansion across anatomical regions and spatial scales.

Imaging of fixed tissue is routine in experimental neuroscience, but is limited by the depth of tissue that can be imaged using conventional methods. Optical clearing of brain tissue using hydrogel-based methods (e.g. CLARITY) allows imaging of large volumes of tissue and is rapidly becoming commonplace in the field. However, these methods suffer from a lack of standardized protocols and validation of the effect they have upon tissue morphology. We present a simple and reliable protocol for tissue clearing along with a quantitative assessment of the effect of tissue clearing upon morphology. Tissue clearing caused tissue swelling (compared to conventional methods), but this swelling was shown to be similar across spatial scales and the variation was within limits acceptable to the field. The results of many studies rely upon an assumption of uniformity in tissue swelling, and by demonstrating this quantitatively, research using these methods can be interpreted more reliably.