Immunostaining of Ventricles and Tracheas

Ventricles were dissected according to Mirzadeh et al., 2010a (link), pre-extracted with 0.1% Triton X in PBS for 1 min, then fixed in 4% paraformaldehyde (PFA) or ice cold methanol for at least 24 h at 4°C, followed by permeabilization in PBST (0.5% Triton X-100) for 20-min room temperature. Tracheas were dissected and cut longitudinally into two, pre-extracted in for 30 s on ice in PEM (0.1 M PIPES (1,4-Piperazinediethanesulfonic acid disodium salt) pH 6.8, 2 mM EGTA (ethylene glycol tetraacetic acid), 1 mM MgSO₄) prior to fixing in ice cold methanol on ice for at least 24 hr. Ventricles and tracheas were blocked in 10% donkey serum in TBST (0.1% Triton X) or 4% bovine serum albumin (BSA) in PBST (0.25% Triton X-100) for 1 hr at room temperature, then placed cilia layer down in primary antibodies (Supplementary file 3) in 4% BSA PBST (0.25% Tween-20) or 1% donkey serum in TBST (0.1% Triton X) for at least 12 hr. Ventricles and tracheas were washed in PBS 3 × 10 min and secondaries (Supplementary file 4) in 4% BSA in PBST (0.25% Triton X-100) or 1% donkey serum in TBST (0.1% Triton X) were added at 4°C for at least 12 hr. Ventricles and tracheas were washed in PBS 3 × 10 min, and ventricles were mounted on glass bottom dishes (Nest, 801002) in Vectashield (VectorLabs), immobilized with a cell strainer (Greiner Bio-One, 542040). Tracheas were mounted on slides with Prolong Gold.