Rat anti cd31

Biotinylation of mouse 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, 7 K MWCO, (Thermo Fisher Scientific, 89882), 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).

Mice were injected with 10% chloral hydrate and transcardially perfused with ice-cold 0.1 M PBS (pH 7.4) followed by ice-cold 4% PFA (Electron Microscopy Sciences, #15713S). Intact brains were dissected out of the skull and postfixed in 4 % PFA at 4 °C for 18 h. Brains were then cryoprotected in 30% sucrose (Sigma, #S0389), frozen and sliced on a cryostat at 35 μm thickness. Sections were washed with PBS three times and incubated in blocking solution (3% normal donkey serum (Jackson Immuno Research, #017-000-121), 0.3% Triton X-100 (Sigma, T9284) in PBS) for 2 h. Sections were then incubated in primary antibodies (rat anti-CD31, 1:300, Biolegend, #102501; rabbit anti-RFP, 1:300, Rockland, #600-401-379) overnight at 4 °C. The next day, sections were washed in PBS with 0.3% Tween-20 (PBST, [Sigma, P7949]) three times for 15 min each, then incubated with anti-rabbit-Cy2 and anti-rat-Cy5 secondary antibodies for 2 h (1:400, Jackson Immunoresearch, #711-225-152, #712-175-153, respectively). Sections were washed again three times with PBST. Slices were then mounted on slides, air-dried overnight, dehydrated, and coverslipped with DPX (Electron Microscopy Sciences, #13510). All slices were imaged using a Zeiss LSM 780 confocal microscope. 3D reconstruction was performed with the IMARIS software (v9.9) (Oxford Instruments Group).