Dfc300

Formalin‐fixed inflated lung sections obtained from unchallenged Cc16^−/− and WT mice that were 6, 12, or 18 months of age were immunostained for two cyclin‐dependent kinases inhibitors (CDKI), p16 and p21, which are markers of cellular senescence (Wu et al. 2016). The sections were incubated with blocking buffer followed by rabbit anti‐p16 IgG, rat anti‐p21 IgG (Abcam, Cambridge, MA), nonimmune rabbit IgG (Dako North America Inc, Carpinteria, CA), or nonimmune rat IgG (Sigma‐Aldrich, St. Louis, MO). The sections were washed in PBS and then incubated with Alexa‐488‐conjugated goat antirabbit F(ab’)₂ or goat antirat F(ab’)₂ (Life Technology, Grand Island, NY). Images of the lung sections were captured using an epifluorescence microscope and a digital camera (Leica DFC300, Allendale, NJ). The numbers of alveolar septal cells that were positively stained for p16 or p21 were counted, and normalized to unit area of alveolar wall measured in pixels (Laucho‐Contreras et al. 2015aa) using MetaMorph software (Molecular Devices, Sunnyvale, CA).

Photomicrographs were obtained the Leica DMLB microscope connected to a Leica DFC 300 digital camera and Leica Applications Suite 4.1 for Windows.

Postnatal day 18 (P18) and P21 old mice were terminally sedated using a 1 : 2:1 solution of fentanyl citrate and fluanisone anaesthesia (Hypnorm, VetaPharma), ddH₂O and midazolam hydrochloride (Hypnovel, Roche) at a dosage of 5 µl per 1 g. After sedation occurred, the tympanic membrane was carefully punctured and mice were injected with a 1% aqueous solution of the fluorescent tracer Fluorescein (Sigma) into both middle ear cavities and left in a prone position. Mice were culled by cervical dislocation after a validated time of 5 min. To visualize the soft palate and nasopharynx to pharynx orifice, the mandible and tongue were dissected and photographed in a supine position to allow a ventral view of the palate using a dissection microscope (Leica MZFiii) and Leica DFC300 camera with fluorescence.