Supplementary Figure 5: Protein epitope preservation after ScaleS and CUBIC treatment

Immunohistochemistry on sections restored from ScaleS (left) and CUBIC (middle) compared with control sections stored in PBS(–) (right). DG, dentate gyrus; GCL, granule cell layer; MF, mossy fiber; SO, stratum oriens; SR, stratum radiatum. Scale bars: 100 μm. See Online Methods for details of 2D-IHC. Cytoskeleton proteins, such as MAP2 and GFAP, were well immunolocalized in the sections prepared from ScaleS- and CUBIC-treated samples. However, substantial differences in synaptic proteins were noted between the ScaleS- and CUBIC-treated samples. ScaleS treatment preserved the immunostaining of presynaptic and postsynaptic proteins enriched in the mossy fiber (MF) and CA3 regions, respectively. As a result, the signal contrast between the MF and CA3 regions was clearly distinguished. By contrast, CUBIC treatment attenuated the intensity and/or specificity of the immunostaining. In addition, the immunostaining of a cell adhesion molecule (PSA-NCAM) in granule cells in the dentate gyrus and MF regions was preserved in the ScaleS-treated sample but not the CUBIC-treated one. The experiment was performed in triplicate for each using different mouse brains.