Ephb2

Immunohistochemistry (IHC) was performed using the primary antibodies, EphB1, EphB2 (Abcam, Cambridge MA), ephrinB1, ephrinB2 (R&D Systems, Minneapolis MN), and ephrinA3 (Antibodies Onlines, Atlanta GA) and tissue microarrays (TMA) comprised of 60 MB. Negative and positive controls were normal fetal cerebellum and tumor tissues expressing the corresponding Eph/ephrins, including breast carcinoma (Abcam, Cambridge MA), respectively. Incubation with anti-Eph or -ephrin (1:150 dilution) was performed overnight at 4°C and immunodetection was performed using the Elite Vectastain ABC system (Vector Laboratories, Burlingame, CA). Color visualization was performed using 3, 3′-diaminobenzide as the chromagen substrate (Innovex Biosciences, Pinole, CA). Haematoxylin was used as the counterstain. Each tissue sample was independently scored for positivity by two neuropathologists (ER or MS). Scoring was performed blinded and the immunostaining results were graded as either negative or positive. The grading definitions used were established by the neurophathologists based on the relative diffuse cellular homogeneity observed for the specific target staining tested.

Western blot of whole cell lysates was performed using the primary antibodies, EphB1, EphB2, ephrinB1, ephrinB2, phospho-EphB1/B2 (Abcam, Cambridge MA), phospho-Src and GADPH (Cell Signaling Technology, Danvers, MA) and goat or rabbit anti-mouse horseradish peroxidase secondary antibodies (Santa Cruz, CA). Each blot is representative of at least three separate experiments.