AlkDala (Alkyne-Functionalized D-Alanine) Labeling

Labeling of live bacteria using an alkyne functionalized D-alanine (alkDala) biorthogonal probe (Siegrist et al., 2013; Shieh et al., 2014) was adapted for use on the murine ocular surface. Enucleated eyes were incubated in a solution of alkDala (10 mM) in Dulbecco’s Modified Eagle Medium (DMEM) at 37°C for 2 h. In other experiments, eyes of IL-1R (-/-) and MyD88 (-/-) mice were first incubated in an antibiotic cocktail of gentamicin (300 μg/mL), ofloxacin (300 μg/mL) and vancomycin (5 mg/mL) in DMEM. After alkDala incubation, all eyes were transferred to pre-chilled 70% EtOH and fixed for 20 min at -20°C. After rinsing, eyes were permeabilized in PBS with Triton-X100 (0.5%) for 10 min with shaking at room temperature (RT), then washed 3 times for 5 min each in PBS with Triton-X100 (0.1%) and BSA (3%) with shaking at RT. Eyes were then transferred to the click-labeling cocktail [in PBS, TBTA (100 μM), CuSO₄ (1 mM), sodium ascorbate (2 mM), 488 nm azide-fluorophore (10 μM), BSA (0.1 mg/mL)] for 1 h with shaking at RT.