Rabbit anti fgf2

Whole telencephalon of E12.5 brains were dissected and lysed for protein with RIPA lysis buffer (Sigma) on ice for 30 min. The lysate is then resolved on a 4–12% Bis-Tris gel (Invitrogen) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with blocking solution (Li-Cor) and incubated with primary antibody rabbit anti Fgf2 (Santa Cruz) diluted 1/500 in blocking solution at 4°C overnight. Mouse anti β-actin antibody (Abcam) diluted at 1/2,500 in blocking solution was included as protein loading control. Bound antibodies were detected by incubation for 45 min with goat anti-rabbit IgG IRDye800CW (Li-Cor) and goat anti-mouse IgG Alexa Fluor 680 (Invitrogen), both 1/12,000. All washes were with 0.1%Tween20 in PBS. Membranes were dried before scanning using an Odyssey infrared imaging system (Li-Cor). Fgf2 signal was normalized to β-actin detected in the same sample.
Control for Fgf2 antibody specificity was performed by combining rabbit anti Fgf2 antibody (Santa Cruz) with a 5-fold, by weight, excess of Fgf2 blocking peptide (Santa Cruz), made up to 500μl with PBS and incubated for 2hr. The reaction was diluted in 50% LiCor/PBS-Tween blocking buffer for incubation with Western blots. Student’s t-test was used for statistical comparison with n = 3.