Clarifying Tissue Clearing.

Methods suitable for Immunolabelling A number of the techniques above have been shown to be compatible with immunolabelling protocols ( table 1 ). It should be noted however that immunolabelling steps should be performed prior to immersing samples in highly viscous final clearing solutions (e.g. FocusClear, CUBIC2, RIMs, high molarity sugar solutions, etc.) as the diffusion of antibodies will be hindered in these conditions. Additionally, very long incubation times (weeks/months) are required for antibodies to fully penetrate through large tissues. For this reason, protocols that embed tissues in large pore hydrogels are more likely to show rapid and complete immunolabelling ( Yang et al., 2014 ). The use of single domain antibodies (reviewed in Holliger and Hudson, 2005 ) or nucleic acid aptamers (reviewed in Bunka and Stockley, 2006 ) which are a fraction of the size of standard antibodies can better access epitopes deep in a sample in a shorter period of time. Additionally, direct fluorophore conjugation to these probes reduces the need for two antibody incubation steps. Finally, a new method termed eTANGO, utilizes dynamic electric fields to move antibodies through large cleared tissue and ensure an even distribution of the label both spatially and temporally across the sample. Using this technique, an entire mouse brain can be stained within hours (KH Chung, personal communication).