Hyperhydratation

These common tissue clearing methods are based on hydrophilic substances, and they expand the tissue volume. They are commonly used for heart tissue clearing as they preserve GFP fluorescence and are compatible with immunostaining. The SCALE is in primarily an aqueous solution of urea, which is compatible with immunostaining (Azaripour et al., 2016) and preserves the GFP signal (Hama et al., 2015). Several improvements of the SCALE method are available: ScaleS, ScaleA2. However, only few studies have used SCALE on the heart tissue, and details about the difference among SCALE methods on heart tissue have yet to be further explored. SCALE clearing was used to clear superficial layers of the embryonic heart (Kolesova et al., 2016) and to perform single cell tracing and analysis of the whole heart during early cardiac development (Li et al., 2016). A drawback of this method is the long incubation time up to months (Keller and Dodt, 2012), or with shorter incubation, tissue is not cleared in its whole depth (Kolesova et al., 2016). CUBIC is a modification of SCALE. It contains only nontoxic water-soluble chemicals (Tainaka et al., 2014); however, sale of one of its components (Quadrol) is monitored as it is a component for explosive fabrication and therefore can be challenging to obtain (authors' experience). The method has been commonly used for heart tissue clearing (Kolesova et al., 2016; Nehrhoff et al., 2016; Yokoyama et al., 2017). The main advantage of CUBIC over the other clearing methods is that CUBIC constituents (Quadrol) effectively elute endogenous chromophores (mainly heme) and thus reduce myocardial and blood autofluorescence (Susaki et al., 2014; Tainaka et al., 2014). The CUBIC tissue clearing protocol can be used for embryonic and adult mouse hearts (Kolesova et al., 2016) and enables one to observe in detail structures such as coronary arteries, fine structure of the ventricular trabeculae, and pectinate muscles. According to the original description, CUBIC is also suitable for tissue clearing after immunolabeling and preserves the fluorescent antibody signal (Tainaka et al., 2014). The main disadvantage is still a long clearing period for larger samples. This significant limitation was recently overcome with a modification named SUT (Wang et al., 2018). SUT is an effective method for clearing and imaging of cardiac microstructures in whole hearts from different species. Wang et al. (2018) accelerated the protocol even further by using electrophoresis to shorten the time of antibody penetration.