Biotin Labeling of Recombinant MMP8 for Brain Imaging

Biotinylation of mouse recombinant MMP8 (rMMP8) (Bio-techne, #2904-MP-010) was performed using the EZ-Link^™ Sulfo-NHS-Biotin kit according to the manufacturer’s instructions (Thermo Fisher Scientific, #A39256). Biotinylated rMMP8 was separated from unbound biotin using Pierce^™ C18 Spin Columns, 7K MWCO, (Thermo Fisher Scientific, #89870), which recovers proteins and macromolecules larger than 7 kDa. Biotinylated rMMP8 was injected retro-orbitally into anaesthetized mice. After 2 h of circulation, mice were euthanized and perfused with ice-cold PBS followed by 4% PFA. Brain tissue processing and imaging was performed as described in the Immunohistochemistry and confocal microscopy section, with the following antibodies: Biotin was visualized using the Oregon Green^® 488 conjugate of NeutrAvidin^® biotin-binding protein (Thermo Fisher Scientific, #A6374). Counterstaining was performed using rabbit anti-NeuN (1:500, Abcam, #ab177487) and rat anti-CD31 (1:300, Biolegend, #102501).