Anti e2a n 649

Human ductal in situ carcinoma (DCIS) samples were obtained through the Biological Resource Center of the Centre Léon Bérard with the agreement of the ethical review board of the Centre Léon Bérard. Samples were used with the patient’s written informed consent. The present study was approved by the ethical review board of the Centre Léon Bérard. DCIS samples were selected on the basis of TWIST1 protein detection, as previously reported [7] (link).
After deparaffinization and rehydratation, tissue sections were boiled in a 10-mM pH 6 citrate buffer for 40 minutes. After saturating unspecific binding sites for 20 minutes with a 1% BSA PBS buffer, sections were incubated overnight with the mouse monoclonal anti-TWIST1 Twist2C1a (Abcam) and the rabbit polyclonal anti-E2A N-649 (Santa Cruz) antibodies in a 0.5% BSA in PBS buffer. Incubation with PLA probes, ligation, and amplification steps were performed according to the manufacturer instructions (Duolink In Situ kit, Sigma). The specificity of the signal was confirmed using paraffin-embedded MDA-MB-436, Hs578T (TWIST1 positive, E12 positive), and HMEC-hTERT (TWIST1 negative, E12 positive) mammary cells in which TWIST1 or TCF3 (the two E2A protein variants E12 and E47 encoding gene) were specifically knocked down through RNA interference. Number of dots per cell was assessed using the ImageJ software.