Fig. 4

IHC staining for pulmonary neuroendocrine cells (PNECs), PGP 9.5, and GRP in influenza-infected mice. Lung sections from Fig. 3 were stained in a mock-infected, c PR8 + vehicle, and e PR8 + NSC77427 for neural/neuroendocrine-specific PGP 9.5, or in b mock-infected, d PR8 + vehicle, and f PR8 + NSC77427 for GRP, a neuroendocrine granule marker. Scale bars shown are 50 μm for a–d, and 10 μm for e and f. Arrows are positioned in each panel within a treatment group to indicate groups of positive cells, except in b, where the arrow indicates GRP− cells adjacent to the PGP 9.5+ cells shown in a. g Graphic image analysis of PGP 9.5+ and GRP+ cells after IHC staining. Slides were blinded by an observer with no knowledge of experimental groups. PCP9.5+ and GRP+ cells were counted throughout each complete lung lobe cross-section for each animal, with validation by ×40 micrographs of each slide. Total tissue area was determined by using ImageI thresholding analysis (methyl green counterstain) because PNECs are mainly observed in alveolar ducts and small bronchioles. *p = 0.009; **p = 0.001