Tx 100

Trachea were excised from newborn piglets and immediately fixed in 4% paraformaldehyde (EMS) in PBS for 1 hr at room temperature. Tissues were then placed in 30% sucrose and incubated overnight at 4°C, followed by quick-freezing in OCT using a dry ice/EtOH bath and stored at −80°C. Prior to immunocytochemistry, frozen blocks of tissue were cryosectioned at 7 μm followed by permeabilization in 0.3% TX-100 (Thermo-Fisher) in PBS for 20 min, and blocked in Super-Block (Thermo-Fisher) with 5% normal goat serum (Jackson ImmunoResearch) for 1 hr, all at room temperature. Tissue sections were then incubated for 2 hr at 37°C with indicated antibodies: β-tubulin IV(1:300, Biogenex), MUC5AC (1:5000, Novus Biologicals), MUC5B (1:2000, Santa Cruz). Sections were then incubated for 1 hr with secondary antibodies goat-anti-mouse Alexa-Fluor-488 and goat anti-rabbit Alexa-Fluor-555 (1:1000, Molecular Probes/Invitrogen) and phalloidin-633 (1:300, Molecular Probes/Invitrogen). Slides were imaged on an Olympus Fluoview FV3000 confocal microscope with a Plan.ApoN 60X oil lens. Images were post-processed using the Olympus imaging software, CellSens.

The standard BCA protein assay was utilized with modifications to accommodate the low protein yield from exosome preparations. Briefly, 5 μl of 10% TX-100 (Thermo Scientific) were added to an aliquot of 50 μl of purified exosomes and incubated 10 min at room temperature. A working ratio of 1:11 was used and incubated in a 96 well plate for 1 h at 37 °C. Absorbance at 562 nm was then measured (SpectraMax Plus 384) and protein concentration estimated from a quartic model fit to the BSA standard curve.

Cells rinsed in phosphate-buffered saline (PBS) were mechanically lifted, harvested, and lysed in SLB + 1% LMNG or Triton X-100 (TX-100, Fischer Scientific), as described above. Lysates were clarified by centrifugation (17,000 x g, 30 min.) and pre-cleared using CL-4B Sepharose beads (50 µL of 50:50 slurry, Pharmacia/GE), with subsequent affinity- and immuno-purifications carried out using the resulting lysates. Beads were washed thrice in SLB and subsequently resuspended in 2 x Laemmli buffer + 20 mM DTT (10 min, 56°C) after the final wash, separated by SDS-PAGE and transferred to PVDF membrane for western blotting. Western blots were performed by incubating membranes in PBST blocking buffer (PBS + 1% Tween-20 supplemented with 5% non-fat dry milk), with subsequent primary and secondary antibody incubations in PBST + 5% non-fat dry milk. Secondary antibodies conjugated with horseradish peroxidase (HRP) were used to detect proteins bound to primary antibodies for enhanced chemiluminescence (ECL) with images captured either on X-ray film (FujiFilm, SuperRX) or by CCD camera (Chemidoc, BioRad) for quantification.