Bx53 dsu

To analyze mucociliary clearance, adult mouse tracheal samples were isolated from wild-type and keratin8-KO mice and observed using a fluorescence microscope and a DSU microscope (BX53-DSU; Olympus). The flow of fluorescent beads (a 500-fold dilution of Fluoresbrite, 0.5 µm; Polysciences, Inc., Warrington, PA, USA) in the mouse trachea was recorded at 25 ms/frame using a water immersion objective lens (LUMPlan FLN 60×; NA 1.00; WD, 2.0 mm; Olympus), an ORCA-Flash 4.0v2 sCMOS camera (Hamamatsu Photonics), and a ThermoPlate (37 °C; Tokai Hit). Hardware was controlled using MetaMorph software (Molecular Devices). After subtracting images processed by Gaussian Blur from all acquired images in ImageJ, each bead was tracked and analyzed using the TrackMate plugin for Imagej2 Fiji.

Cells plated on glass coverslips were fixed in cold methanol at −20°C for 10 min or fixed in 1% formaldehyde in HBS at RT for 8 min. The fixed cells were treated with 0.25% Triton X‐100 in HBS at RT for 5 min and washed three times with HBS. After soaking in HBS containing 1% bovine serum albumin (BSA) at RT for 10 min, the samples were treated with primary antibodies at RT for 60–180 min, followed by washing three times with HBS and incubating with secondary antibodies at RT for 60 min (In some experiments, rhodamine‐phalloidin [#R415; Molecular Probes] was added to detect F‐actin). The samples were washed three times with HBS, shortly soaked in Milli‐Q water (Millipore), and mounted with Faramount Mounting Medium (#S3025; DAKO). Immunofluorescent micrographs were acquired using a fluorescence microscope (BX51 or BX53; Olympus), a fluorescence microscope with a disk scanning unit system (BX53‐DSU; Olympus), or a Spinning Disk Confocal Super Resolution Microscope (SD‐OSR; Olympus).