Quantitative assessment of alternative CLARITY procedures in mouse brain

Krolewski DM, Kumar V, Martin B, Tomer R, Deisseroth K, Akil H, Watson SJ Jr
Society for Neuroscience. 2015.


The CLARITY technique for visualizing 3-dimensional fluorescence immunohistochemistry (IHC) requires prior polymerization of a brain-infused acrylamide fixative to generate a hydrogel mesh through which lipid is subsequently removed with sodium dodecyl sulfate (SDS) detergent (Chung et al., 2013). The resulting linkage between hydrogel monomers, formaldehyde, and proteins using the original protocol proved challenging for achieving sufficient lipid clearing and antibody penetration in transparent tissue. More recently, these obstacles have been tempered by reducing and increasing acrylamide and SDS concentrations, respectively (Tomer et al., 2014; Yang et al., 2014). However, the degree of neuronal protein preservation related to such condition changes is still not completely understood. To address this, our laboratory has developed previously unpublished alternative acrylamide formulas and performed IHC experiments in passively cleared brain samples. Using both standard confocal microscopy and a CLARITY Optimized Light Sheet Microscopy (COLM) system, we employed multiple software packages and conducted quantitative analyses of neuronal and non-neuronal markers. The results of the present study will serve to further optimize CLARITY brain transparency methods.