Immunofluorescence Staining of Ciliary Ultrastructure

Respiratory epithelial cells on glass slides, stored at -80°C, were defrosted, washed with PBS and fixed by incubating with 4% PFA for 15 min. The samples were then treated with 0.2% Triton-X 100 for 10 min. to permeabilize the cell membranes, and incubated with 1% skim milk at 4°C overnight to block unspecific binding of the antibodies. Prepared samples were incubated with the primary antibodies for 2–3 h at room temperature, washed and incubated with the cross-absorbed secondary antibodies for 25 min at room temperature, and then with Hoechst 33342 (Sigma). The primary antibodies were IgGs directed against markers of various elements of the ciliary ultrastructure: acetylated α-tubulin (mouse monoclonal, SIGMA); α/β-tubulin (rabbit polyclonal, Cell Signaling Technology); DNAH5 (mouse monoclonal, generated [10 (link)]); DNALI1 (rabbit polyclonal, AVIVA); LRRC6 (rabbit polyclonal, AVIVA); CCDC39 (rabbit polyclonal, SIGMA). Immunofluorescence images were taken with Olympus Bx41 Microscope and processed with Isis software Metasystem International) and Adobe Creative Suite 4.