3 3 diaminobenzidene

Lungs were inflated and fixed with 10% formalin in PBS, then embedded in paraffin as described [10 (link)]. Tissue blocks were cut into 5-μm sections, dewaxed with xylene and ethanol, washed with PBS, exposed to 3% H₂O₂ in 90% methanol for 30 min, then washed with PBS containing 0.05% Tween-20 (PBST) and antigens were retrieved in a heated pressure cooker for 10 min in 10 mM sodium citrate, pH 6.0. Specimens were blocked with 10% (v/v) goat serum in PBS for 1 h, washed with PBST and then labelled for 1 h with anti-SNAP23 serum raised against the peptide DRIDIANARAKKLIDS, Synaptic Systems) diluted 1:500 in PBS. Slides were then washed with PBST and incubated for 30 min at 21°C with horseradish peroxidase (HRP)-labelled goat anti-rabbit antibodies (Millipore) diluted 1:1000 in PBST, washed in PBST, developed with 3,3′-diaminobenzidene (Vector Laboratories) and counterstained with haematoxylin. For co-localization of SNAP23 with airway epithelial lineage markers, tissue blocks were cut into 2-μm sections, then stained with anti-SNAP23 serum as above; adjacent slices were stained with goat antibodies against club cell secretory protein (CCSP; 1:10000, gift of Franceso DeMayo, Baylor College of Medicine, Houston, U.S.A.) or mouse monoclonal antibodies against acetylated α-tubulin (1:200, Sigma–Aldrich) and developed with HRP-labelled secondary antibodies as above.