Multiscale 3-dimensional pathology findings of COVID-19 diseased lung using high-resolution cleared tissue microscopy

Guang Li; Sharon E. Fox; Brian Summa; Carola Wenk; Aibek Akmatbekov; Jack L. Harbert; Richard S. Vander Heide; J. Quincy Brown

The copyright holder for this preprint (which was not peer-reviewed) is the . Sample preparation for three-dimensional (3D) imaging. After fixation the sample was rinsed overnight in PBST solution made from phosphate-buffered saline (PBS, Sigma) with 0.2% Triton-X 100 (Sigma), and then stained and cleared following the protocol of Reder et al. 7 Briefly, the sample was immersed in a 1:1000 v/v solution of TO-PRO 3 iodide (T3605, Thermo Scientific) in PBST overnight with light shaking. Afterwards, the sample was dehydrated in graded solutions of 0% (deionized water), 25%, 50%, 75% and 100% ethanol solution with deionized water (1 hour each), followed by a final 1 hour wash in fresh 100% ethanol. The dehydrated sample was stained with alcoholic Eosin solution (Surgipath 3801615, Leica Biosystems) diluted in 1:1,000 v/v ratio with 100% ethanol overnight, then rinsed in ethyl cinnamate (ECi, Sigma) twice for 1 hour each time to undergo tissue clearing and refractive index matching. 9 Finally, the sample was incubated in fresh ECi and degassed under vacuum for 1 hour.