Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA

Schrom, Eva; Huber, Maja; Aneja, Manish; Dohmen, Christian; Emrich, Daniela; Geiger, Johannes; Hasenpusch, Günther; Herrmann-Janson, Annika; Kretzschmann, Verena; Mykhailyk, Olga; Pasewald, Tamara; Oak, Prajakta; Hilgendorff, Anne; Wohlleber, Dirk; Hoymann, Heinz-Gerd; Schaudien, Dirk; Plank, Christian; Rudolph, Carsten; Kubisch-Dohmen, Rebekka

Cells were cultured on coverslips in six-well plates. After washing with PBS, 20 mg/mL tetramethylrhodamine-conjugated wheat germ agglutinin (Thermo Fisher Scientific) in Hank's balanced salt solution (Gibco/Life Technologies) was added to the samples and incubated for 10 min in the incubator. Samples were washed with PBS and fixed in 4% paraformaldehyde for 10 min at RT. Cells were washed again and incubated for 10 min with 0.2% Triton X-100 at RT. After another washing step, cells were blocked in PBS supplemented with 10% FCS and 0.05% Triton X-100 for 30 min at RT. Slides were then incubated for 1 hr with primary antibody against ACE2 (5 mg/mL, AF933; R&D Systems) at RT, followed by another washing step. Secondary anti-goat AF488 antibody (1:400, A11087; Thermo Fisher Scientific) and DAPI (1 mg/mL; Sigma-Aldrich) were added and incubated for 1 hr at RT. After a final wash, slides were mounted in FluorSave (Merck Chemicals) and viewed under a Leica DMi8 microscope (Leica Microsystems).