For generation of monoclonal antibodies against mouse P2Y12R, a Lou/c rat was immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 40 µg ovalbumin-coupled peptide corresponding to P2Y12R aa 325-342 TSGTNKKKGQEGGEPS (Peps4LS, Heidelberg) dissolved in 400 µl PBS containing 5 nmol CpG2003 (TIB MOLBIOL, Berlin, Germany), and 400 µl Incomplete Freund's adjuvant (Sigma-Aldrich). After 8 weeks, a boost without Freund's adjuvant was given i.p. and s.c. 3 days before fusion. Fusion of mouse immune spleen cells with the myeloma cell line P3X63-Ag8.653 was performed using polyethylene glycol 1500 according to standard procedure . After fusion, the cells were plated in 96-well plates in RPMI 1640 medium supplemented with 20% fetal calf serum, 1mM pyruvate, 1x non-essential amino acids and HAT media supplement (Hybri-Max, Sigma-Aldrich). Hybridoma supernatants were screened 10 days later in a flow cytometry assay (iQue, Intellicyt; Sartorius) using biotinylated peptides captured on streptavidin beads (PolyAN, Berlin) and incubated for 90 min with hybridoma supernatant and Atto-488-coupled isotypespecific monoclonal mouse-anti-rat IgG secondary antibodies. Antibody binding was analyzed using ForeCyt software (Sartorius). Positive supernatants were further validated in capture ELISA, Western blot and immunohistochemistry. Hybridoma cells from selected supernatants were subcloned by limiting dilution to obtain stable monoclonal cells lines. Experiments in this work were performed with clone P2YM 17A12 (ratIgG2c/ƙ).