The enzymatic activity of MMP-3 was determined using a fluorescence resonance energy transfer (FRET) peptide and immunocapture assay as previously described elsewhere with minor modifications.17 (link) Briefly, 50 µg total protein of macrovascular or brain homogenates were incubated at 4°C for 2 h with rabbit polyclonal anti-MMP-3 antibody (Cat.No. sc-6839-R; Santa Cruz Biotechnology, Dallas, TX, USA). A/G agarose beads were then added and allowed to incubate overnight at 4°C. The beads were then washed and samples were transferred to black 96 well plate and 100 ul of 2mM 5-FAM/QXL™520 FRET peptide (Cat. No. 60580-01; AnaSpec, San Jose, CA, USA) in assay buffer were added per well. Plates were incubated for 8 h at 37°C, then relative fluorescence units (RFUs) were read and monitored at excitation/emission wavelengths of 485/528 nm in a Synergy HT Multi-mode microplate fluorescence reader (BioTek, Winooski, VT, USA) running Gen5™ data analysis software.