Figure 4.

Characterization of the availability of sialylated glycans and their distribution between cell types

(A) Lectin staining (red) of human lung and ALI culture cryosections performed in parallel. Representative images are shown; nuclei are stained blue (DAPI); Aw, airway; scale bars represent 100 μm. Cryosections pre-treated using Sialidase from Arthrobacter ureafaciens (Sialidase A) to cleave sialic acid were also stained in parallel to confirm staining is specific to sialylated glycans (see Figure S2 for representative images, together with staining of murine lung cryosections). (B and C) Dual lectin staining (red) and immunohistochemistry (green) of cryosections of B-ALI (B) and MucilAir (C) cultures using cell-type-specific antibodies for basal (cytokeratin 5), ciliated (β-tubulin), goblet (mucin 5AC), and club cells (CC10). Cells labeled by immunohistochemistry that co-localize with lectin staining appear yellow (white arrows); nuclei are stained blue (DAPI); scale bars represent 50 μm. To confirm absence of SNA-stained basal and ciliated cells in B-ALI cultures, images were captured using a Zeiss LSM 780 inverted confocal microscope (63× oil immersion) to achieve maximum resolution (B). Stained MucilAir culture cryosections were imaged using a widefield fluorescence microscope (EVOS FL Auto 2) (C).