Rabbit anti tmem119

Coronal sections were air-dried, washed with 1X PBS, blocked with 2% cold water fish skin gelatin (Sigma, Inc., St. Louis, MO, USA) in 0.2% triton-X100 for 1 h, incubated with a primary antibody diluted in blocking solution overnight at room temperature (RT), and then washed with 1X PBS six times 10 min each and incubated with appropriate secondary antibody solution (1:250 dilution) for 1 h at RT. Slides were then washed with 1X PBS and then mounted with media-containing DAPI counterstain (SouthernBiotech, Birmingham, AL, USA). Antibodies used are goat anti-EphA4 at 1:100 dilution (R&D systems, Minneapolis, MN, USA), rabbit anti-TMEM119 at 1:100 dilution (Abcam, Waltham, MA, USA), rabbit anti-CCR2 at 1:100 dilution (Abcam, Waltham, MA, USA), rat anti-CD68 at 1:100 dilution (Thermofisher, USA), rabbit anti-Iba1 at 1:200 dilution (FUJIFILM Wako Chemicals U.S.A. Corporation), donkey anti-goat 594 (Thermofisher, USA), donkey anti-rabbit 647 (Thermofisher, USA), donkey anti-rabbit 555 (Thermofisher, USA), and donkey anti-rat 647 (Thermofisher, USA). Image acquisition was performed using a Zeiss 880 confocal microscope (Carl-Zeiss, Oberkochen, Germany).

For tissue preparation and immunohistochemistry mice underwent terminal anaesthesia and perfusion fixation with 4% paraformaldehyde. Brains (or slices, following termination of electrophysiological experiments) were placed in fixative at 4 °C for > 48 h and were then washed and sectioned at 20–60 μm. Control mammary tumour and naïve spleen sections were from tissue collected as part of other studies [37 (link)]. The following primary antibodies were used as described previously [40 (link)]: rabbit anti-Iba1 (1:500; Fujifilm Wako Chemical Corporation), mouse anti-human nuclear antigen (1:100; Merck), Alexa Fluor 647-conjugated rat anti-CD45 (1:100; Biolegend), and rabbit anti-TMEM119 (1:100; Abcam). TMEM119 staining required antigen retrieval and slices were incubated at 90 °C for 15 min in 10 mM Citrate Buffer (pH 6) containing 0.05% Tween-20. The secondary antibodies were Alexa Fluor 488/633-conjugated goat anti-rabbit/mouse, as appropriate (1:500; Molecular Probes). Sections were counter-stained with Hoechst 33258 and mounted in VectaShield (Vector Laboratories) or Prolong Gold (Molecular Probes). Images were acquired using a Zeiss AxioObserver Z1 microscope fitted with W Plan Apochromat 20x/1.0 NA and 63x oil/1.0 NA objective lenses and LSM 880 laser scanner. Images were acquired sequentially and saved for offline processing using Zeiss Zen2 software.

The mouse brain tissues were fixed with formalin and embedded in paraffin. The counterstaining of hematoxylin was conducted. The standard method of peroxidase-conjugated streptavidin and the substrate 3,3’-diaminobenzidine (DAB) were used sequentially. Immunostaining was carried out with rabbit anti-Netrin-1 antibody (Abcam) or rabbit anti-PHF1 antibody (Abcam) or rabbit anti-TMEM119 (Abcam). PHF-1 positive neuronal cells were quantified for assessing neuronal cell loss. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of mouse Netrin-1 (ELM-NTN1; RayBiotech), mouse DCC (ELM-DCC; RayBiotech), mouse UNC5a (abx552620, Abbexa Ltd, Cambridge, UK), mouse UNC5b (abx552623, Abbexa Ltd), mouse UNC5c (abx520698, Abbexa Ltd), mouse IL-1β (ab197742; Abcam), mouse A2B adenosine receptor (A2B; STJE0006946, St John’s Laboratory, London, UK), mouse IL-6 (M6000B; R&D System), mouse tumor necrosis factor alpha (TNFα, ab208348; Abcam), mouse interferon gamma (IFNɣ, ab282874; Abcam), mouse IL-10 (M1000B; R&D System), and mouse arginase 1 (ARG1, ab269541; Abcam). Specific kits were utilized as per the manufacturer’s instructions.

Mice were transcardially perfused with PBS followed by 4% paraformaldehyde in PBS. Mouse brains were post-fixed for 6 h or overnight at 4 °C and processed for frozen sectioning as previously detailed^{34 (link)}. Free floating 50-µm cryosections and 14-µm cryosections on slides were prepared from Microfetti and lineage negative barcoded BM chimeras, respectively. Tissues were permeabilized in blocking solution (0.1% Triton-X-100, 5% bovine albumin, and PBS) for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies: 1:500 rabbit anti-Iba-1 (Wako), 1:200 goat anti-Iba-1 (Novus), 1:1000 chicken anti-GFP against eBFP (Abcam), 1:1000 rabbit anti-TMEM119 (Abcam), 1:500 rabbit anti-P2RY12 (Ana Spec), 1:200 goat anti-APOE (Millipore), and 1:100 mouse anti-MHC Class II (Abcam). Antigen retrieval was performed prior to APOE staining for 40 min at 92 °C in pH 9 citrate buffer. Corresponding secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 647 (Life Technologies) were applied at 1:1000 with nuclear counterstain by 4′,6-diamidino-2-phenylindole (DAPI, Sigma) at 1:5000 for 2 h at room temperature. Sections were mounted in ProLong® Diamond Antifade Mountant (Life Technologies).