Exosome samples solubilized in Laemmli buffer were loaded onto SDS-PAGE gels by equal volume or equal particle number[18 (link)], separated electrophoretically and transferred to nitrocellulose. Membranes were blocked with 10% fat-free milk/water for 30min at room temp on a rocking platform. Blocking solution was exchanged with primary antibody solutions (antibodies diluted in 5% fat-free milk/TBST) and incubated on a rocking platform for 4hrs at room temperature or overnight at 4°C. Primary antibodies used were as follows: anti-Fn (#sc8422, Santa Cruz), anti-annexin A2 (#610068, BD Transduction Laboratories), anti-annexin A6 (#ab52221, Abcam), lactadherin (MFG-E8)(#sc271574, Santa Cruz), myocilin (custom polyclonal [27 (link)]). Primary antibodies were removed and blots were washed 3 times, for 5 minutes with TBST. Antibody solutions containing secondary, HRP-conjugated antibodies (goat anti-mouse HRP # 115035146, goat anti-rabbit HRP # 111035144, Jackson ImmunoResearch, West Grove, PA) were added and incubated for 1hr at room temperature while rocking. Blots were then washed 3 times for 5 min in TBST and developed using chemiluminescent reagents (HyGLO; #E2400, Denville Scientific, South Plainfield, NJ) and X-ray film (#30–101, Genesee Scientific, San Diego, CA).
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