E0433

Indirect ELISA was performed to determine total anti-mCD99 antibody levels. Blood samples were coagulated overnight at 4°C and centrifuged twice at 7,000 rpm for 10 min at 4°C in a microcentrifuge. The supernatant (serum) was stored at −20°C until use. Volumes used per well in ELISA were 50 μl, unless indicated otherwise. 96-well ELISA plates (Nunc A/S, Roskilde, Denmark) were coated with 2 μg/ml mCD99 protein and then blocked with 100% horse serum (100 μl/well) (Sigma-Aldrich), both for 1 h at 37°C. After a single wash with PBS (B. Braun Medical, Oss, The Netherlands) for 1 min, the plates were incubated with serum of TRXtr-mCD99 or TRXtr-vaccinated mice for 45 min at 37°C, diluted 1:10 in 100% horse serum, which was further diluted 1:15 in 10% Rosetta Gami extract (final serum dilution 1:150) to reduce non-specific binding of the serum. Thereafter, plates were incubated with biotinylated polyclonal goat anti-mouse Ig (E0433, Dako Cytomation) for 45 min and streptavidin-horseradish peroxidase (Strep-HRP) (Dako Cytomation) for 30 min, both diluted 1:2,000 in 0.01% PBS-T at 37°C. After each incubation step, plates were washed four times with PBS. HRP activity was detected with TMB substrate (T-8665, Sigma-Aldrich) and absorbance was measured at 655 nm after 15 min using a Biotek Synergy HT microplate reader (Biotek).

For immunostainings on mouse brain sections, 5 µm sections were prepared from the paraffin embedded samples. After paraffin removal, samples were treated with citrate buffer (S2031; DAKO) followed by blocking with 5% BSA after washing with PBS. Next, sections were incubated ON with the primary antibodies anti-Alix (1:1000; ab76608; abcam), anti-Flotillin1 (FLOT1) (1:600; ab41927; abcam), anti-CD63 (1:200; sc-31214; Santa Cruz biotechnology), anti-RAB5 (1:500; ab18211; Abcam), anti-AnnexinA2 (ANXA2) (1:200; ab54771; Abcam) and anti-C3 (1:100; PA5-21349; Thermo Scientific). After a washing step, sections were incubated with the secondary antibodies goat anti-rabbit biotin (1:500; E0432; DAKO) or goat anti-mouse biotin (1:500; E0433; DAKO) for 2 h at RT. Next, amplification of the signal was performed using the ABC system (PK-6100; Vector laboratories) and TSA (SAT700001EA; Perkin Elmer) according to manufacturer’s instructions and samples were incubated with streptavidin-DyLight 633. Finally, the samples were counterstained with Hoechst (1 µg/ml) and the sections were mounted using 2% n-propyl gallate. A Leica TCS SP5 II confocal microscope or a Zeiss LSM780 confocal microscope was used for imaging. 3D reconstructions of z-stacks were created with Volocity.