3D imaging is becoming more and more popular, as it allows us to identify interactions between structures in organs. Furthermore, it gives the possibility to quantify and size these structures. To allow 3D imaging, the tissue sample has to be transparent. This is usually achieved by using optical tissue clearing protocols. Although using optical tissue clearing often results in perfect 3D images, these protocols have some pitfalls, like long duration of sample preparation (up to several weeks), use of toxic substances, damage to antibody staining, fluorescent proteins or dyes, high refractive indices, and high costs of sample processing.Recently we described [Huang et al., Scientific Reports 9(1): 521 (2019)] a fast, safe, and inexpensive ethyl cinnamate (ECi) based optical tissue clearing protocol. Here, we present extensions of our protocol with respect to the deparaffinization of old paraffin‐embedded samples allowing 3D imaging of the blocks. In addition, we learned to remove ECi from the samples allowing the use of routine immunolabeling protocols. Furthermore, we demonstrate new pictures of lungs after expansion microscopy and adaptation of already existing protocols. The aim of our work is, in summary, to describe the advances in these methodologies, focusing on the morphological imaging of kidneys and lungs.

Purpose: 3D imaging of the lung is not a trivial undertaking as during preparation the lung may collapse. Also serial sections and scans followed by 3D reconstruction may lead to artifacts. The present study aims to figure out the best way to perform 3D imaging in lung research. Materials and Methods: We applied an optical tissue clearing (OTC) method, which uses ethyl cinnamate (ECi) as a fast, non-toxic and cheap clearing solvent, for 3D imaging of retrograde perfused lungs by laser confocal fluorescence microscopy and light sheet fluorescence microscopy. We also introduced expansion microscopy (ExM), a cutting-edge technique, in 3D imaging of lungs. We examined and compared the usefulness of these techniques for 3D lung imaging. The ExM protocol was further extended to paraffin-embedded lung metastases blocks. Results:The MHI148-PEI labeled lung vasculature was visualized by retrograde perfusion combined with trachea ligation and ECi based OTC. As compared with transcardiac perfusion, the retrograde perfusion results in a better maintenance of lung morphology. 3D structure of alveoli, vascular branches and cilia in lung were revealed by immunofluorescence staining after ExM. 3D distribution of microvasculature and neutrophil cells in 10 years old paraffin-embedded lung metastases were analyzed by ExM. Conclusions: The retrograde perfusion combined with trachea ligation technique could be applied in the lung research in mice. 3D structure of lung vasculature can be visualized by MHI148-PEI perfusion and ECi based OTC in an efficient way. ExM and immunofluorescence staining protocol is highly recommended to perform 3D imaging of fresh fixed lung as well as paraffin-embedded lung blocks.