polyclonal goat anti spp1  (R&D Systems)

Journal: The Journal of Experimental Medicine

Article Title: Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism

doi: 10.1084/jem.20171529

Figure Lengend Snippet: CV-KO-5XFAD mice have increased microglia activation compared with R47H-KO-5XFAD and KO-5XFAD mice. (A) RT-qPCR on whole cortical tissue for neurodegeneration-associated microglial activation markers Cst7, Spp1, and Gpnmb, as well as classical inflammatory cytokines Tnf, Il6, and Il1b, showing dramatically higher microglial activation markers but not inflammatory cytokine transcripts in CV-KO-5XFAD compared with other groups. (B) Microarray analysis of activation markers in sorted microglia shows that KO-5XFAD and R47H-KO-5XFAD cluster together and separately from CV-KO-5XFAD. Gene names and values are given in Table S1. Some variability within CV-KO-5XFAD, R47H-KO-5XFAD, and KO-5XFAD groups was also observed, with females in all groups having higher activation markers. Color scale represents log 2 deviation from the row mean. (C) Spp1 protein is detected by confocal microscopy in CV-KO-5XFAD brains and largely absent from R47H-KO-5XFAD and KO-5XFAD brains. Bar, 50 µm. (D) The percent of Iba-1 + (microglia) pixels that were also Spp1 + was quantified in cortex and hippocampus for all 5XFAD and non-5XFAD groups. ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA with Holm-Sidak multiple comparisons testing. Data are presented as mean ± SEM.

Article Snippet: Floating sections were blocked with 3% BSA and 0.25% Triton X-100 in PBS, and then stained with anti–Iba-1 (rabbit polyclonal, 1:5,000; Wako; or goat polyclonal, 1:1,000; Abcam), anti–human TREM2 ECD (goat polyclonal, 1:500; R&D Systems), anti–human TREM2 C terminus (D814C rabbit mAb, 1:500; Cell Signaling), anti-Spp1 (goat polyclonal, 1:500; R&D Systems), anti-APP (22C11 mouse mAb, 1:1,000; Millipore) and/or anti-NeuN (D3S3I rabbit mAb, 1:500; Cell Signaling) overnight at 4°C followed by staining with anti–rabbit IgG DyLight 549 (1:2,000; Vector), anti–goat IgG Alexa Fluor 488 (1:2,000; Abcam), anti–rabbit IgG Alexa Fluor 647 (goat polyclonal, 1:1,000; Invitrogen), anti–goat IgG-biotin (donkey polyclonal, 1:2,000; Invitrogen), streptavidin Alexa Fluor 647 (1:2,000; Invitrogen), methoxy-X04 (3 µg/ml; Tocris), and/or TO-PRO-3 iodide (300 nM; Thermo Fisher Scientific) for 1 h at room temperature.

Techniques: Activation Assay, Quantitative RT-PCR, Microarray, Confocal Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism

doi: 10.1084/jem.20171529

Figure Lengend Snippet: qPCR primers

Article Snippet: Floating sections were blocked with 3% BSA and 0.25% Triton X-100 in PBS, and then stained with anti–Iba-1 (rabbit polyclonal, 1:5,000; Wako; or goat polyclonal, 1:1,000; Abcam), anti–human TREM2 ECD (goat polyclonal, 1:500; R&D Systems), anti–human TREM2 C terminus (D814C rabbit mAb, 1:500; Cell Signaling), anti-Spp1 (goat polyclonal, 1:500; R&D Systems), anti-APP (22C11 mouse mAb, 1:1,000; Millipore) and/or anti-NeuN (D3S3I rabbit mAb, 1:500; Cell Signaling) overnight at 4°C followed by staining with anti–rabbit IgG DyLight 549 (1:2,000; Vector), anti–goat IgG Alexa Fluor 488 (1:2,000; Abcam), anti–rabbit IgG Alexa Fluor 647 (goat polyclonal, 1:1,000; Invitrogen), anti–goat IgG-biotin (donkey polyclonal, 1:2,000; Invitrogen), streptavidin Alexa Fluor 647 (1:2,000; Invitrogen), methoxy-X04 (3 µg/ml; Tocris), and/or TO-PRO-3 iodide (300 nM; Thermo Fisher Scientific) for 1 h at room temperature.

Techniques: