ALPL protein, human

Brain tissue processing, immunohistochemistry and immunofluorescence experiments were performed as described previously^{37 (link),41 (link)}. Hemibrains were isolated and post-fixed in 4% (w/v) PFA overnight at 4 °C before preservation in 30% (w/v) sucrose in PBS. Hemibrains were sectioned coronally or sagittally at a thickness of 50 μm on a freezing–sliding microtome, and sections were stored in cryoprotective medium at −20 °C. Free-floating sections were blocked with appropriate serum before incubation at 4 °C with primary antibodies at the following concentrations for confocal microscopy: goat monoclonal anti-CD31 (1:100, AF3628, R&D), fluorescein-labelled lectin (1:200, Vector Laboratories), rabbit monoclonal anti-AQP4 (1:500, AB2218, Millipore Sigma), rat anti-CD13 (1:100, MCA2183EL, Bio-Rad), goat anti-ALPL (1:100, AF2909, R&D), mouse anti-NeuN (1:400, MAB377, Millipore), goat anti-albumin (1:100, NB600-41532, Novus), rabbit anti-transferrin (1:100, ab82411, Abcam or 1:100, AF3987, R&D), rabbit anti-collagen I (1:100, ab21286, Abcam), goat anti-Ibal (1:500, ab5076, Abcam), rabbit anti-MFSD2A (1:300, gift from C. Gu), rat anti-TFRC (1:100, Novus, NB100-64979) and rabbit anti-phospho-STAT3 (1:100, 9145S, CST). Sections were washed, stained with Alexa Fluor-conjugated secondary antibodies (1:250), mounted and coverslipped with ProLong Gold (Life Technologies) before imaging on a confocal laser scanning microscope (Zeiss LSM880). Age-related autofluorescence was quenched with 1 mM CuSO₄ in 50 mM ammonium acetate buffer (pH 5), as previously described^{42 (link)}. National Institutes of Health ImageJ software was used to quantify the percentage of vasculature (CD31 or AQP4) covered by mIgG, MFSD2A, TFRC, CD13, AQP4 or ALPL, as described previously^{18 (link)}. All analyses were performed by a blinded observer. Alizarin red staining was performed as described previously^{21 (link)}, with minor adaptations: sections were incubated for 1 h in 40 mM Alizarin red in PBS (pH 7.4) at room temperature, and extensively washed overnight with PBS before mounting. Images of brain sections were acquired by conventional light microscopy to detect calcified nodules. Sections with biotinylated plasma were blocked overnight in 6% BSA at room temperature, detected with streptavidin-Alexa Fluor 647 (1:1,500, Thermo Fisher) for 2 h and washed overnight before mounting. Sections containing L-azidohomoalanine-labelled plasma were blocked overnight in 6% BSA at room temperature, incubated in 45 mM iodoacetamide (Millipore Sigma) in 100% methanol for 1 h, washed, detected with 1.2 μM sDIBO (Thermo Fisher Scientific) in 100% methanol, and washed overnight before mounting. Vascular ALPL activity was measured using the Red Alkaline Phosphatase Substrate Kit (SK-5100, Vector Laboratories) with 20-min incubation. Risedronate-647 (RIS-647, BioVinc, BV500101) was either injected intravenously (retro-orbital) at 8 nmol per mouse and brains were collected 18 h later, or brain sections were stained with 0.1 nM RIS-647 for 2 h at room temperature alongside fluorescein-labelled lectin (1:200, Vector Laboratories).