Thunder imaging system

Whole slide scans (ZEISS Axioscan 7) were performed on the histology-stained samples for histopathological analysis for all studies. The histology stain images presented in Fig. 1 were obtained on a Lecia DMi-8 microscope equipped with a Leica DFC7000T camera and the fluorescent IF images presented in Figs. 1 and 2 were obtained with the Leica Thunder imaging system. IF images related to Figs. 3 and 4 were obtained with a Zeis Axioscan 7 slide scanner.
Quantification of ACTA2 expression by IF for data presented in Figs. 1 and 2 was on 11–18 10× fields of view obtained on the Lecia Thunder imaging system. ImageJ was used to apply systematic thresholding and quantify the area of positive signal. For Fig. 3, whole tissue section scans (ZEISS Axioscan 7) of ACTA2 expression were quantified using the HALO IF module, and the AI module was used to remove large airways and airway and vessel lumen regions.

Microtissues were stained with Calcein-AM and propidium iodide (PI) dual staining to ensure viability. Calcein AM is used to evaluate cell viability, as it is only converted into its green-fluorescent form, Calcein, by active metabolism within a living cell. PI is a fluorescent stain interacting with nucleic acids. A healthy, intact cell membrane would inhibit the cellular uptake of PI and therefore prevent staining of live cells. Briefly, 1 ml of fresh culture medium containing 1 μl Calcein-AM (10 μg/μl) (Invitrogen, Waltham, Massachusetts, United States), 1 μl PI (10 μg/μl) (Sigma, St. Louis, Missouri, United States), and 1 μl Hoechst (10 μg/μl) (Thermo Fisher, Waltham, Massachusetts, United States) was added to the microtissues. The samples were incubated at 37°C and 5% CO₂ for half an hour. The supernatant was removed, and the microtissues were washed two times with PBS, and fixed with 4% paraformaldehyde (PFA) for 15 min. After two additional PBS washing steps the microtissues were transferred to a glass slide and DAKO mounting media was added. For PI positive control, microtissues were fixed in 4% PFA overnight at 4°C and stained in a similar way. Imaging was done using an inverted microscope (Leica THUNDER Imaging System, Wetzlar Germany).