Broad applicability of a streamlined Ethyl Cinnamate-based clearing procedure

Abstract

Turbidity and opaqueness are inherent properties of tissues which limit the capacity to acquire microscopic images through large tissues. Creating a uniform refractive index, known as tissue clearing, overcomes most of these issues. These methods have enabled researchers to image large and complex 3D structures with unprecedented depth and resolution. However, tissue clearing has been adopted to a limited extent due to a combination of cost, time, complexity of existing methods and potential negative impact on fluorescence signal. Here we describe 2Eci (2nd generation Ethyl cinnamate based clearing method) which can be used to clear a wide range of tissues, including cerebral organoids, Drosophila melanogaster, zebrafish, axolotl, and Xenopus laevis in as little as 1-5 days while preserving a broad range of fluorescence proteins including GFP, mCherry, Brainbow, and alexa-fluorophores. Ethyl cinnamate is non-toxic and can easily be used in multi-user microscope facilities. This method will open up clearing to a much broader group of researchers, due to its broad applicability, ease of use, and non-toxic nature of Ethyl cinnamate.Summary statementThe non-toxic, broadly applicable, and simplified protocol of 2Eci tissue clearing makes it possible for non-specialist labs to use clearing approaches on conventional inverted microscopes.

Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling

“…CLARITY is ideal to visualize endogenous fluorescence of reporters such as GFP, which is partially quenched by solvent-based clearing (e.g., ethyl cinnamate) ( Klingberg et al, 2017 ; Puelles et al, 2017 ). However, such reporters can also be visualized by antibody labeling, given the stability of fluorescent secondary antibodies and modified solvent-based protocols that preserve endogenous fluorescence signals ( Masselink et al, 2018 ). In contrast to the original ethyl cinnamate protocol ( Klingberg et al, 2017 ), our approach combines a modified ethyl cinnamate optical clearing method and immunolabeling, which uses an antigen-retrieval step to improve antibody binding.…”

Biomedical Applications of Tissue Clearing and Three-Dimensional Imaging in Health and Disease

“…The 3D approach enabled visualization of normal and damaged glomerular capsules and revealed changes in the distribution and morphology of parietal epithelial cells. Recently, comparative studies have been carried out using several reagents and protocols, including Scale and ECI, CLARITY, CUBIC1, and uDISCO, to study crescentic diseases that may include podocyte depletion ( Puelles et al., 2019 ; Masselink et al., 2019 ). Related to pathologies in the kidney, potassium-mediated tubule remodeling has also been revealed by optical clearing ( Saritas et al., 2018 ) and ethanol-ECi (ethyl-cinnamate) cleared kidneys have enabled the study of renal nephrosis.…”

“…Human cerebral organoids have been cleared with 2ECi (second-generation ethyl cinnamate-based clearing method), which was developed to overcome long clearing times of aqueous-based clearing methods and fluorescence quenching of organic solvents ( Masselink et al., 2019 ).…”

A 3D atlas of the dynamic and regional variation of pancreatic innervation in diabetes

“…In addition, we applied a novel alternative approach to visualize islet vasculature by combining insulin immunolabeling with fluorophore-tagged dextran or CD31 antibody, followed by optical clearing using ethyl cinnamate (ECi) to preserve fluorescence while allowing for additional immunostaining tissue ( fig. S4) (41). These data demonstrate that modification of optical clearing protocols allows for visualization of multiple markers in pancreatic tissue.…”