On-slide clearing and imaging of 100-µm-thick histological sections using ethyl cinnamate and epifluorescence

Abstract

Introduction: Thick histological samples are difficult to image without proper tissue clearing methods. Among these methods ethyl cinnamate (ECi)-based clearing preserves antigenicity and is compatible with immunofluorescent labeling. In contrast to many other clearing protocols, ECi-based clearing is fast and is done as a final step after standard immunofluorescent labeling protocols. Aim: We aimed to develop a simple method of ECi-based tissue clearing approach of thick (100 µm) sections attached to a slide glass. Materials and methods: Samples of human colorectal cancer underwent fixation and frozen section. We used indirect immunofluorescence to label Von-Willebrand factor, which is expressed in blood vessels. After completion of labeling and nuclear DAPI staining, the material was dehydrated using alcohols. Finally, the material was cleared and mounted with ECi and subsequently visualized via standard widefield epifluorescence microscopy. Image analysis of z-stacks were evaluated for the depth of visible signals and compared them to non-cleared samples. Results: Compared to the non-cleared sections, the ECi-cleared sections provided a 2.5-fold increase in the observable tissue thickness following immunofluorescent staining. Further, the proposed approach is time efficient (4 days from tissue preparation to final imaging) as compared to other tissue clearing methods and low cost as it minimizes the use of large amounts of reagents. Conclusion: ECi-based clearing is a very effective simple extension of standard immunofluorescent protocols and can be implemented in future experiments.