Anti mouse igg

Primary antibodies specific for HBV proteins included anti-HBV core protein mouse monoclonal antibody (MAb) 8C9-11 (72 (link)), anti-HBV core protein mouse MAb sc-23945 (Santa Cruz), both recognizing linear epitopes within the HBV core protein, and HBV capsid/capsid-intermediate-specific rabbit polyclonal antibody (PAb) B0586 (Dako). Primary antibodies used for epitope tags and cellular targets included anti-HA rat MAb 3F10 (Core Facility for Monoclonal Antibodies, Helmholtz Zentrum München), mouse MAb against the His₆ tag (Clontech), anti-PML protein mouse MAb sc-966 (Santa Cruz), polyclonal rabbit anti-PML protein PAb NB100-59787 (Novus Biologicals), polyclonal rabbit anti-PML protein ab72137 (Abcam), monoclonal mouse anti-SUMO2/3 ab81371 (Abcam), and anti-beta-actin mouse MAb AC-15 (Sigma-Aldrich). Secondary antibodies conjugated to Alexa Fluor 488 were purchased from Thermo Scientific, and secondary antibodies coupled to horseradish peroxidase or Alexa Fluor 647 were anti-rabbit IgG, anti-mouse IgG, anti-mouse light chain IgG, and anti-rat IgG (all from Dianova).

HMC 1.2 or EoL-1 cells (5 × 10⁵/ml) were incubated for 1 h on ice with sCD30, ultracentrifugation-enriched CD30-containing EVs or HBSS buffer alone. After washing with PBS containing 1% albumin and 0.1% sodium azide, cells were incubated for another 30 min on ice with FITC-coupled SGN-35 (0.1 μg/ml). After washing in the above PBS, cells were evaluated by flow cytometry. Vesicles alone were incubated overnight with polybead carboxylate microspheres (4.5 μm; Polysciences INC, Warrington, PA). The beads were blocked with 1% BSA (v/w) in PBS. Then, aliquots were incubated with unlabeled or fluorescence-labeled antibodies. Aliquots with unlabeled antibody were in a second step labeled with fluorescence-labeled anti-mouse IgG (Dianova, Hamburg, Germany). Beads were evaluated by flow cytometry.