Streptavidin conjugated horseradish peroxidase hrp

Half-volume 96 well plates (3690; Corning) were incubated with 50 μl per well of the VU-1D9 anti-human EpCAM antibody (0.5 μg·ml⁻¹ in PBS; Invitrogen) for 12 h at 4°C, then washed with PBS and blocked with 5% bovine serum albumin (BSA) in PBS supplemented with 0.01% Tween^® 20 (PBST, pH 7.0; Sigma-Aldrich) for 2 h. These plates were then aspirated and incubated for 12 h at 4°C with EV samples diluted in PBS (25 μl per well) to concentrations of 1.2, 0.8, 0.5, 0.4, and 0.2 µg/µl. Plates were then washed with PBST and incubated for 1 h at 37°C with 50 μl per well of biotinylated MEM-61 anti-human CD9 antibody (0.5 μg.ml⁻¹; Invitrogen) suspended in 5% BSA/PBST. Sample wells were then washed five times with PBST and incubated for 1 h at 37°C with 50 μl streptavidin conjugated horseradish peroxidase (HRP, 1:5000 dilution, Cell Signaling Technology) suspended in 5% BSA/PBST. Plates were then washed five times with PBST and incubated for 15 min at 37°C with 50 μl per well of 3,3’,5,5’-Tetramethylbenzidine reagent (eBioscience Inc.), then supplemented with 50 μl per well of 2 M H₂SO₄ stop solution and analyzed for absorbance at 450 nm. The EV ELISA standard curve was calculated using GraphPad Prism 8.0.2 software (GraphPad Software) plotting optical density versus EV concentration.

For fluorescent immunohistochemistry, primary antibody specificities and dilutions used are summarized in S1 Table. Secondary antibodies from ThermoFisher Scientific were diluted 1:500 and were either Alexa Fluor 488 goat anti-rabbit, streptavidin-conjugated Alexa Fluor 568, or streptavidin-conjugated Alexa Fluor 594.
For western blot analysis, the primary antibody was either the anti-hamster PrP mouse monoclonal antibody 3F4 conjugated to biotin (1:10,000 dilution), 3F4 derived in-house from hybridoma supernatant (1:1,000 dilution), or the anti-mouse PrP antibody 6D11 (1:5,000 dilution). Secondary antibodies were either streptavidin-conjugated horse radish peroxidase (HRP, Cell Signaling Technologies) diluted 1:250,000, or sheep anti-mouse IgG conjugated to horse radish peroxidase (GE Healthcare) diluted at 1:100,000.