To identify virus-positive cells, cells on chamber slides and Transwell inserts, human intestinoids, slides of human small intestine, or mouse tissues were subjected to immunofluorescence staining, as described previously (45 (link)). Briefly, after permeabilization and blocking, cells, intestinoids, and tissues were stained with a guinea pig antiserum against MERS-CoV NP, followed with secondary antibodies including goat anti–guinea pig Alexa Fluor 488 immunoglobulin G (IgG) or goat anti–guinea pig Alexa Fluor 594 IgG (Abcam). For intestine explants and intestinoids, CK19 (YM3051, ImmunoWay), a marker of intestinal epithelial cell, was costained with an anti-NP antibody. To define the identity of the viral NP-positive cells in the tissues of infected hDPP4 mice, apart from the labeling of MERS-CoV NP, double staining was performed using a rat anti-mouse monoclonal CD68 antibody (FA-11, Abcam) and Alexa Fluor 568 goat anti-rat IgG (Life Technologies). After staining, intestinoids were whole-mounted with VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories). The whole-mounted intestinoids, chamber slides, Transwell inserts, and tissue slides were imaged using a Carl Zeiss LSM 780 or 800 confocal microscope.