Immunocytochemical Staining of VCD-OOCs

Immunocytochemical staining for α-smooth muscle actin (14-9760-82, Thermo Fisher Scientific, Waltham, MA) for hFM-DEC, cytokeratin(CK)-14 (ab51054: Abcam for ECTO, Cambridge, MA, USA), CK-18 (ab668; Abcam) and MUC5A (ab3649; Abcam) for ENDO, pan-cytokeratin (#4545, Cell Signaling Technology, Danvers, MA) for VEC, type I collagen (ab34710; Abcam) and vimentin (ab92547; Abcam) STROMA were performed after 48 h, as previously described.^{49 (link),57} Manufacturer’s instructions were used to calculate the staining dilutions to ensure uniform staining. After 48 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X, and blocked with 3% bovine serum albumin in 1× PBS before incubation with primary antibodies overnight at 4 °C. After washing with 1× PBS, the VCD-OOCs were incubated with Alexa Fluor 488–, 594, and 647–conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1:400 in 1× PBS for 2 h in the dark. The VCD-OOCs were washed with 1× PBS and treated with NucBlue^® Fixed ReadyProbes Reagent (R37606; Thermo Fisher Scientific) to stain the nucleus.