Quantitative Analysis of Apical Mucin Secretion

Apical mucin secretion was analyzed semi-quantitatively. For each 3D model, three different randomly chosen cross sections were evaluated by estimating the amount of immunofluorescence signals of Muc5AC and Muc5B, respectively, per total surface of a cross section. Scoring was: zero points (no detection), one point (sporadic detection on the surface), two points (<50% of the surface), three points (>50% of the surface). (For Muc5B: six models each medium; for Muc5AC: six models with AECG medium, five models for PC ALI medium, all from four donors). The presence of intraepithelial mucus cysts was analyzed using the same cross sections, which were used for the mucin score. Immunofluorescence signals of Muc5AC and Muc5B within the epithelial layer in DAPI-free areas were counted as presence of intraepithelial mucus cysts. Given is the percentage of cross sections on which intraepithelial immunofluorescence signals of either Muc5AC or Muc5B were detected.