Alexa fluor couple secondary antibodies

Both uninfected and infected h3ACs were fixed in 4% paraformaldehyde (PFA) for 3 hr at 4°C, and then dehydrated in PBS with 30% sucrose (v/v) (Sigma-Aldrich). The h3ACs were embedded with optimal cutting temperature (OCT) compound (Leica) and cut into 7-10 μm thick sections. The sections were blocked with PBS with 5% normal donkey serum and 1% Triton X-100 (Sigma-Aldrich). The sections were incubated with primary antibodies overnight at 4°C, then washed, and incubated with host matched Alexa Fluor-couple secondary antibodies (1:1000, Jackson ImmunoResearch Laboratories) for 1.5 hr at RT. Following DAPI incubation, slides were mounted. E-cadherin (1:300, R&D SYSTEMS, AF748), NP (1:200, Sino Biological, 40143-MM05), NP (1:200, Sino Biological, 40143-T62), dsRNA (1:2, SCICONS, 10030005), ACE2 (1:400, abcam, ab15348), TMPRSS2 (1:400, Santa Cruz, 515727), pro-SFTPC (1:400, abcam, ab90716), MX1 (1:400, GeneTex, GTX110256), and HTII-280 (1:50, Terrace Biotech, TB-27AHT2) were used.

Attached hAT1-like cells were fixed with 4% PFA at 4°C for 3 hr. After cells were blocked in PBS with 5% normal donkey serum and 1% Triton X-100 (Sigma-Aldrich), sections were incubated with primary antibodies overnight at 4°C, then washed, and incubated with host matched Alexa Fluor-couple secondary antibodies (1:1000, Jackson ImmunoResearch Laboratories) (1:1000) for 1.5 hr at RT. Following DAPI incubation, slides were mounted. AGER (1:400, R&D SYSTEMS, AF1145), Aquaporin5 (1:200, abcam, ab92320) pro-SFTPC (1:400, abcam, ab90716) were used.

To check polarity of h3ACs, we conducted Z stack whole mount imunofluorescnce. H3ACs were seeded with the Phenol-free Matrigel (Corning) in the 8-well chamber slides (Thremo Fisher Scientific). Each well was fixed with 4% PFA (Biosesang) at 4°C for 45 min and washed with PBS for 5 min 2 times. Organoids were blocked with PBS with 1% BSA (Sigma-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich). The sections were incubated with primary antibodies overnight at 4°C, then washed, and incubated with host matched Alexa Fluor-couple secondary antibodies (1:1000, Jackson ImmunoResearch Laboratories) for 1.5 hr at RT. Alexa 647 Phalloidin (1:100) were incubated for 1hr at RT. Following DAPI incubation, slides were mounted. Crb3 (1:400, NOVUS BIOLOGICALS, NBP1-81185), HTII-280 (1:200, Terrace Biotech, TB-27AHT2) were used.