Goat anti ang 1

Heart tissue samples from anterior or basolateral left ventricular walls were obtained from explanted hearts after transplantation and were snap frozen within less than 4 h. Frozen samples were then oriented, and 6 µ-thick cryostat sections were cut and immunostained with a panel of primary antibodies applied in TRIS-buffered saline and with the use of appropriate secondary antibodies and fast-red substrate. The following panel of primary antibodies was used: goat anti angiogenin, Santa Cruz, sc-1408 (1:150); goat anti Ang-1, Santa Cruz, sc-6319 (1:200); goat anti Ang-2, Santa Cruz, sc-7016 (1:200); rabbit anti Tie-2, Santa Cruz, sc-9026 (1:100); goat anti procollagen-I, Santa Cruz, sc-8782 (1:50). Human tonsil or nasal polyps were used as a positive control. For the negative control slides, normal goat or rabbit nonspecific immunoglobulins (Santa Cruz Biotechnology) were used.

Commercially available antibodies were obtained as follows: rabbit anti-mouse α-smooth muscle actin (α-SMA), rabbit anti-mouse collagen I, rat anti-mouse F4/80 (CI:A3-1), rabbit-anti-mouse CD31 antibodies were from Abcam (Cambridge, England), rabbit anti-mouse phospho-Smad 2/3(D27F4) and Smad2/3 (D7G7), rabbit anti-mouse GAPDH (D16H11) antibodies were from Cell Signaling (Danvers, Massachusetts), goat anti-Ang-1 was from Santa Cruz (Santa Cruz, CA), and horseradish peroxidase (HRP)-conjugated anti-mouse β actin antibody was from Sigma Aldrich. Alexa 594-conjugated secondary antibody was purchased from Molecular Probes (Carlsbad, CA). HRP-conjugated anti-rabbit and anti-goat secondary antibodies were from Santa Cruz. Histochoice was purchased from Amresco (Solon, OH).