Tissue sections (4 µm thick) were cut from FFPE tissue blocks and mounted onto glass slides. Slide-tissue sections were baked at 70 °C for 1 h and subsequently soaked in xylene (Thermo Fisher Scientific) for 10 min × 3 rounds. Rehydration and HIER of tissue sections were performed in a similar manner as described above except that rehydrating washes were done for 180 s each. After HIER and cooling, slides were then washed twice with Milli-Q water (Millipore Sigma) subjected to a 10-min protease digestion at 40 °C with Protease III (322337, Bio-Techne) diluted to 1:20 in 1X PBS. Slides were then washed for 2 × 2 min Milli-Q water before a 15-min H2O2 block at 40 °C (322335, Bio-Techne). Slides were then washed for 2 × 2 min Milli-Q water before an overnight hybridization at 40 °C with probes against the human ACE2 mRNA (848151, Bio-Techne) or SARS-CoV-2 Spike mRNA (848561, Bio-Techne). Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences). Slides were then processed as described above for IF IHC staining for anti-MUC5AC (Abcam ab212636), ACE2 (Abcam ab15348), and/or cytokeratin 8 (Santa Cruz sc-8020) staining. Fluorescent images were acquired and processed as detailed above.
Lee I.T., Nakayama T., Wu C.T., Goltsev Y., Jiang S., Gall P.A., Liao C.K., Shih L.C., Schürch C.M., McIlwain D.R., Chu P., Borchard N.A., Zarabanda D., Dholakia S.S., Yang A., Kim D., Chen H., Kanie T., Lin C.D., Tsai M.H., Phillips K.M., Kim R., Overdevest J.B., Tyler M.A., Yan C.H., Lin C.F., Lin Y.T., Bau D.T., Tsay G.J., Patel Z.M., Tsou Y.A., Tzankov A., Matter M.S., Tai C.J., Yeh T.H., Hwang P.H., Nolan G.P., Nayak J.V, & Jackson P.K. (2020). ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs. Nature Communications, 11, 5453.
+ Open protocol