Tmem119

Primary microglia were isolated from P0–3 C57BL/6 or RAGE KO pups using the shaking method, adapted from previously described protocols^{37 (link)}. Briefly, P0 litters were euthanized via decapitation, and brains were removed and collected under aseptic conditions. Brain tissue was dissociated to a single cell suspension using the neural tissue dissociation kit (Miltenyi Biotec), and the resulting cell suspension was centrifuged at 400g for 10 minutes. The cells were then resuspended in complete DMEM (with 4.5 g/L glucose) containing 10% FBS, 1% penicillin-streptomycin, and 0.5 ng/ml recombinant mouse GM-CSF (R&D Biosystems, Minneapolis, USA) before being filtered through a 70 μm cell strainer (431751, Corning). Cell suspensions from each litter of pups were plated into a different 175 cm² flask and cultured until confluence (7–10 days). Microglia were recovered from culture by manual shaking. Growth medium containing ‘shaken’ microglia was centrifuged at 400g for 10 minutes, then counted and plated alone in a 48-well plate at 200,000 cells per well. Flow cytometry analysis using antibodies against CD45 (Clone 30-F11, BioLegend), CD11b (Clone M1–70, BD Biosciences), and TMEM119 (Clone 106–6, Abcam) indicated a microglial purity of greater than 95%.

Brian tissue sections were incubated overnight with primary antibodies at 4°C and then with corresponding fluorochrome-conjugated secondary antibodies at room temperature for 1 h. The following primary antibodies were used: anti-human NKp46 (195314; R&D Systems), CD49a (SR84; BD Bioscience), CD57 (MA1-81071; Invitrogen), CD68 (ED1; Abcam), CD4 (H-370; Santa Cruz), CD8 (UCH-T4; Santa Cruz), CD19 (HIB19; BioLegend), CD16b (CLB-gran11.5; BD Bioscience), CD66b (polyclonal; Bioss), perforin (dG9; BioLegend), CD69 (D-3; Santa Cruz), Caspase-3 (9661; CST), CD31 (JC/70A; Abcam); anti-mouse NKp46 (M20; Santa Cruz), CD49a (Ha31/8; BD Bioscience), ly6G (1A8; BioLegend), TMEM119 (28-3; Abcam), CD68 (ED1; Abcam), CD8 (53-6.7; eBioscience), CD4 (GK1.5; eBioscience), CD19 (1D3; BD Bioscience), CD31 (polyclonal; Abcam), claudin5 (4C3C2; Invitrogen), and ZO-1 (ZO-1-1A12; Invitrogen). The following fluorochrome-conjugated secondary antibodies were used: donkey anti-rabbit 488 (1:1,000; Invitrogen), donkey anti-rabbit 546 (1:1,000; Invitrogen), donkey anti-goat 546 (1:1,000; Invitrogen), donkey anti-mouse 594 (1:1,000; Invitrogen), donkey anti-mouse 488 (1:1,000; Invitrogen), goat anti-rat 488 (1:1,000; Invitrogen), and goat anti-rat 555 (1:1,000; Invitrogen). Images were acquired on a fluorescence microscope (Olympus BX-61).

Immunolabeling‐enabled three‐dimensional imaging of solvent‐cleared organs (iDISCO) was performed using the readily available protocol at https://idisco.info/idisco-protocol/ with slight modifications. In brief, samples were fixed with 4% PFA and prepared using the methanol pretreatment; samples were then incubated in permeabilization solution for 1 day followed by blocking solution for 1 day. Samples were incubated with the primary antibodies anti GFP (Abcam; 1:200; chicken), human CD11b (Abcam; 1:200; mouse), and TMEM119 (Abcam; 1:200; rabbit) and with secondary antibodies Cy3, Alexa Fluor 488, or Alexa Fluor 647 for anti‐chicken, goat, mouse, or rabbit IgG antibodies (all from Jackson ImmunoResearch Laboratories; 1:200); incubation was set to 2 days with washing steps between for 1 day. Succeeding clearing steps were done following the recommended times in the protocol. Samples where then mounted with PO‐PRO‐1 Iodide (Invitrogen) mixed with DABCO and imaged using a Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

Mouse brains were harvested and dissociated, and leukocytes were isolated and blocked with the antibody CD16/CD32 (Clone 93; BioLegend) to prevent nonspecific binding as previously described [29 (link)] before staining with antibodies against mouse leukocyte antigens, CD45 (Clone 30F11; BD Biosciences), CD11b (Clone M1/70; BD Biosciences), Tmem119 (Clone 106-6; Abcam), MHC-II (Clone I-A/I-E; BD Biosciences), CD86 (Clone GL-1; BioLegend), Ly6G (Clone 1A8; BD Biosciences), CD3 (Clone 145-2C11; BD Biosciences), or CD68 (Clone FA-11; BioLegend). For detection of cell viability, cells were stained with the fixable viability dye (Invitrogen) for 30 min on ice in the dark. For detection of Ki67⁺ cells, cells were fixed and permeabilized by Cytofix/Cytoper (BD Biosciences) before incubation with the anti-Ki67 antibody (Clone 16A8; BioLegend). The stained cells were analyzed by the flow cytometer, CantoII (BD Biosciences) and analyzed with the FlowJo software (Treestar Inc.).

Cells were washed in FACS buffer (1% BSA, 0.1% sodium azide in PBS), then incubated in FACS buffer containing functional viability dye (65-0866-14, ThermoFisher Scientific) along with anti-CD45 (1:80, BioLegend, clone 30-F11), anti-CD11b (1:200, BD Biosciences, clone M1–70) and anti-transmembrane protein 119 (Tmem119; 1:500, Abcam, clone 106-6) antibodies for 15 min at 4 °C in the dark. After staining, cells were washed with FACS buffer, and flow cytometry was performed using the BD LSRFortessa™ Cell Analyze. Data analysis was conducted using FlowJo.

After fixing with an ice‐cold paraformaldehyde 4% solution diluted in a 0.1 M potassium phosphate‐buffered solution (4% paraformaldehyde [PFA]) for 30 minutes at room temperature, cells were permeabilized with 0.25% Triton X‐100 for 15 minutes at room temperature. The cells were then blocked using donkey serum (1:20) for 30 minutes at room temperature before incubating with primary antibody for 2 hours and then with secondary donkey antibody for 1 hour coupled to either Cy3, Alexa Fluor 488, or Alexa Fluor 647 for anti‐chicken, goat, mouse, or rabbit IgG antibodies (all from Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania; 1:200). Between each step the cells were washed with PBS three times for 5 minutes. Primary antibodies used were against the human CD11b (Abcam, Cambridge, Massachusetts; 1:200; rabbit), human ionized calcium‐binding adapter molecule 1 (Iba1; Abcam; 1:200; goat), human transmembrane protein 119 (TMEM119; Abcam; 1:200; rabbit), fractalkine or chemokine ligand 1 (CX3CR1; Abcam; 1:200; mouse), human CD33 (Abcam; 1:200; mouse), major histocompatibility class II HLA‐DR (Abcam; 1:200; mouse), and green fluorescent protein (GFP; Abcam; 1:200; chicken). As negative control the primary antibody was omitted. All antibodies used in the present work are listed in Table S6A.