Rabbit anti fgf2 antibody

Gene expression in tumor and HUVEC cells was examined by quantitative real-time RT-PCR as described previously [8 , 26 (link)]. Sequences of specific primers used for RT-PCR were as the following: 5′-GAATTGCGTCATTTAAAGCCTAG-3′ and 5′-GTTTCATCCTACCACTCCCAATT-3′ for MALAT1; 5′-CCGTTACCTGGCTATGAAGG-3′ and 5′-ACTGCCCAGTTCGTTTCAGT-3′ for FGF2; and 5′-AGGCCAACCGCGAGAAG-3′ and 5′-ACAGCCTGGATAGCAACGTACA-3′ for β-actin. For the analysis of protein expression by immunoblot, cells were lysed, protein extracted and separated by gel electrophoresis. After western transfer, membranes were probed with rabbit anti-FGF2 antibody (1:250) (Santa Cruz Biotech, Santa Cruz, CA), followed by horseradish peroxidase-conjugated anti-rabbit (1:10,000) antiserum (Santa Cruz Biotech, Santa Cruz, CA). Protein bands were visualized with SuperSignal (Pierce, Rockford, IL). The membranes were lastly re-probed with a mouse anti-actin antibody (Sigma) as loading controls.

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.