Multiple MMPs are present in the collagen implants after incubation on the CAM.

Embryos containing the collagen implants were incubated for 66 hours before harvesting. Twenty-micron cryosections through bFGF/VEGF-treated collagen implants were stained using affinity-purified MMP-specific antibodies (red) and counterstained for nuclei (green). (A) Chicken MMP-2 appears in a pericellular pattern within the extracellular matrix. Bar indicates 200 μm. Inset, bar indicates 20 μm. (B) The chicken MMP-9–like enzyme is associated with both PMNs and the extracellular matrix. Bar indicates 200 μm. In the inset, individual positively stained PMNs are enmeshed in a positively staining matrix (asterisks). Bar indicates 10 μm. The affinity-purified polyclonal antibodies were subjected to immunodepletion using purified antigens prior to staining to demonstrate the specificity of the MMP-2 (C) or MMP-9 (D) signals. For depletion of antibodies, purified MMP-2, or MMP-9, was electrophoresed and transferred to nitrocellulose. The nitrocellulose strips were blocked with serum and incubated overnight with dilute solutions of affinity-purified anti–MMP-2 or anti–MMP-9 antibody. The adsorbed antibody solutions were used for immunostaining and yielded no signal (C, D).