Proposal for a simple and easy-to-implement protocol for three-dimensional tissue imaging that is compatible with observation using a confocal microscope

Tissue observation has traditionally been limited to obtaining two-dimensional information from thinly sliced tissues due to issues with light transmission and antibody penetration. In recent years, three-dimensional tissue observation methods combining tissue clearing and deep immunostaining methods have been reported. However, due to the significantly different procedures in these methods from conventional immunostaining methods and the requirement for an expensive and specialized light-sheet microscope for tissue observation, the widespread adoption of these methods has been limited. To promote the shift from the current two-dimensional tissue observation to three-dimensional tissue observation using a combination of tissue clearing and immunostaining, it is essential to establish a simple and easy-to-implement protocol that is compatible with observation using a confocal microscope, which is available in many facilities. In this study, we first examined the effects of tissue clearing and staining conditions of immunostaining with thin tissue slices. We showed that CUBIC-L enhances immunolabeling without diminishing the immunoreactivity of antigens. We also showed that high detergent concentrations enhance the intensity of immunoreactivity and that a two-step staining procedure is suitable for our proposed protocol. Based on the results, we propose a simple protocol that can be easily adapted from conventional methods and is compatible with confocal microscopes. The results of this study are expected to facilitate a shift from traditional methods to three-dimensional tissue observation techniques that combine tissue clearing and immunostaining, contributing to the broader adoption of three-dimensional tissue observation.