Modeling of fibrotic lung disease using 3D organoids derived from human pluripotent stem cells

For RNAscope in situ hybridization, 8-µm FFPE tissue sections from HPS and IPF patients were incubated with probes specific for IL11 (ACD, 425281), SFPTC (ACD, 452561-C2), and EpCAM (ACD, 310281-C3) after proper treatment of the slides with H2O2, target retrieval, and protease digestion according to manufacture’s instructions of the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, 323100). Opal Multiplex IHC Kit (PerkinElmer, NEL811001KT) was used to develop RNA signals, using Opal 520 for IL11, Opal 570 for SFPTC, and Opal 690 for EpCAM, respectively.

After counter staining with DAPI, the slides were imaged using the Vectra 3 automated multispectral microscope (PerkinElmer) and the Phenochart 1.0 software (PerkinElmer) at the Human Immune Monitoring Core (HIMC) of Columbia University Irving Medical Center (RRID:SCR_016740). Images were then processed and analyzed in the inForm 2.4.2 software (PerkinElmer) for quantification of signal levels of each target and their location and co-location in the tissue.