Immunofluorescence labeling of Prox-1(−/−) lungs in situ at E14.5 (a, b) and in lungs from E13 after 2 days in explant culture (c–f). (a) Prox-1(−/−) E14.5 lung showing weak diffuse immunostaining for PGP9.5 in the cytoplasm of epithelial cells from a smaller distal airway including PNEC/NEB (green signal), with positive Mash-1 expression (red signal) in nuclei. Inset: higher magnification of a Mash-1-positive cell with background PGP9.5 staining. (b) The Prox-1(−/−) E14.5 lung dual immunostained for Mash-1 (red signal) showing positive NEB cell nuclei, whereas immunolabeling for Prox-1 (green signal) is negative (original magnification × 480). Wild-type (c, d) and Prox-1(−/−) (e, f) embryonic lungs grown in organ culture showing appropriate expression of PNEC/NEB cell cytoplasmic markers (green signal) PGP9.5 (c, e) and CGRP (d, f), whereas their nuclei are Mash-1 (red signal) positive. Arrows point to NEB (original magnification × 400).