Mouse NKp46/NCR1 Antibody

Mouse NKp46/NCR1 Antibody Induces IFN-gamma Secretion in Activated Mouse NK Cells. Mouse NKp46/NCR1 Antigen Affinity-purified Polyclonal Antibody induces IFN-? secretion in mouse natural killer (NK) cells activated with 25 ng/mL Recombinant Mouse IL-2 (Catalog # 402-ML) and 25 ng/mL Recombinant Mouse IL-12 (Catalog # 419-ML), in a dose-dependent manner, as measured using the Quantikine Mouse IFN-? ELISA Kit (Catalog # MIF00). The ED₅₀ for this effect is typically 0.4-2.4 µg/mL.

Detection of NKp46/NCR1 in Mouse DX5/CD49b⁺Splenocytes by Flow Cytometry. Mouse DX5/CD49b⁺splenocytes were stained with Goat Anti-Mouse NKp46/NCR1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2225, filled histogram) or control antibody (Catalog # AB-108-C, open histogram), followed by Allophycocyanin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0108).

NKp46/NCR1 in Mouse Spleen. NKp46/NCR1 was detected in perfusion fixed frozen sections of mouse spleen using Goat Anti-Mouse NKp46/NCR1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2225) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse NKp46/NCR1 by Immunocytochemistry/Immunofluorescence IL-2/Anti-IL-2 Antibody Complex Treatment Triggers NK Cell-Dependent Acute Sensory Loss after Partial Sciatic Nerve Crush(A) Peripheral blood sampled 16 days post-injury in IL-2 complex or IgG control mice. NKp46+DX5+ NK cells (U = 15.00, p = 0.0019); CD3+CD8+ T cells (t = 15.78, p < 0.0001); CD3+CD4+ T cells (t = 9.719, p < 0.0001). Mann-Whitney or Student’s unpaired t test was used.(B) Daily pinprick response. Male wild-type C57BL/6 mice received partial crush of the sciatic nerve on day 0 followed by daily injection of IL-2 complex or IgG control (i.p.) for 4 consecutive days (arrows). Two-way ANOVA was used. Effect of treatment: F(1,315) = 20.69, p < 0.0001. Bonferroni post-test ∗∗p < 0.01 (t = 3.784), ∗∗∗p < 0.001 (t = 4.741).(C) Heatmap showing mean sensitivity to pinprick along the lateral hind paw. Note the broad loss of sensitivity at day 6 in IL-2-complex-treated mice.(D) Area under the curve measurements in IL-2-complex-treated and IgG control mice. Days 1–4: p = 0.9513, t = 0.06181; days 5–10: ∗∗∗p = 0.0083, t = 2.916; days 11–15: p = 0.4354, t = 0.7951. Student’s unpaired t test was used.(E) Effect of IL-2 complex treatment on NKp46-YFP+ cell infiltration to sciatic nerve 6 days after partial crush injury. Sciatic nerve sections (14 μm) were immunolabeled with anti-GFP and beta -tubulin III antibodies. Scale bars, 100 μm. Arrows indicate individual YFP+ cells.(F) Quantification of NKp46-YFP+ cells per square millimeter in images of different regions of the nerve. Two-way ANOVA: effect of IL-2 complex, F(1,16) = 56.31, p < 0.0001; effect of region, F(3,16) = 23.73, p < 0.0001. Bonferroni post-tests: crush, t = 8.062; distal, t = 6.414 ∗∗∗p < 0.001. ns, not significant. n = 3 sections per region, per mouse, per treatment.(G) Photograph of spleens isolated from mice 1 day after final injection of IgG or IL-2 complex.(H) Peripheral blood sampled 16 days post-injury in IL-2-complex-treated wild-type mice, which received either anti-NK1.1 antibody or isotype control. NKp46+DX5+ NK cells (t = 15.37, ∗∗∗p < 0.0001), CD3+CD8+ T cells (U = 22.00, p = 0.1473), and CD3+CD4+ T cells (t = 3.035, p = 0.0079). Student’s unpaired t test was used.(I) Loss of NKp46-YFP+ cells from peripheral blood in anti-NK1.1-antibody-treated mice. Student’s unpaired t test, t = 15.51, ∗∗∗p = 0.0001.(J) Wild-type mice received either anti-NK1.1 to deplete NK cells or isotype control antibody followed by partial crush of the sciatic nerve on day 0. All mice were treated with IL-2 antibody complex (arrows). Two-way ANOVA. Effect of antibody depletion: F(1,240) = 21.21, p < 0.0001. Bonferroni post-test, ∗∗∗p < 0.001 (t = 4.542).(K) Heatmap showing mean sensitivity to pinprick along the lateral hind paw. Note the broad loss of sensitivity at day 6 in isotype control mice.(L) Area under the curve measurements in isotype control and anti-NK1.1-treated mice following IL-2 complex treatment. days 1–4: t = 0.1508, p = 0.8815; days 5–10: t = 2.390, p = 0.0295; days 11–15: t = 1.040, p = 0.3130). Student’s unpaired t test was used.See also Figure S5. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30712871), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse NKp46/NCR1 by Immunocytochemistry/Immunofluorescence Acutely Cultured Embryonic but Not Adult DRG Neurons Reveal Susceptibility to NK-Mediated Cytotoxicity by RAE1(A) Immunolabeling of co-culture (4 h) between embryonic (top) or adult (bottom) DRG neurons ( beta -tubulin, magenta) and either freshly isolated (control) or IL-2-stimulated natural killer (NK) cells (NKp46, green). The inset shows a high-magnification image of NK cell in contact with embryonic DRG neurite.(B) LDH-release cytotoxicity assay of acutely cultured (1 day in vitro) embryonic (top) and adult (bottom) DRG at various Effector (NK):Target (DRG) (E:T) ratios. Matched two-way ANOVA: embryonic DRG, F(1,10) = 100.01, p < 0.0001); adult DRG, F(1,10) = 1.25, p = 0.2982). Three replicate co-cultures for each DRG group.(C) Still images of in vitro time-lapse confocal Ca2+ imaging of rhodamine 3 AM-loaded embryonic (top) and adult (bottom) DRG (magenta) co-cultured with IL-2-stimulated NK cells (green) isolated from adult male NKp46-YFP mice.(D) Frequency histogram (30 s time bins) of neurite Ca2+ events in embryonic (top) and adult (bottom) DRG during NK co-culture. Cumulative area under the curve (right). Student’s paired t test; t = 2.290, p = 0.045. n = 6 fields of view from two repeat co-cultures per group.(E) RT-PCR of mRNA transcripts in freshly isolated splenic NK cells and embryonic and adult DRG.(F) qRT-PCR shows higher Raet1 mRNA expression in embryonic compared to adult DRG tissue. Student’s paired t test; t = 16.16, p < 0.0001. n = 5 mice, or replicates per group.(G) Western blot of embryonic and adult mouse DRG tissue (40 μg loading) with pan-RAE1 antibody and beta -actin control. Images are representative of three independent experiments.(H) Selective siRNA knockdown reduces RAE1 protein (top) and Raet1 mRNA (bottom) expression in embryonic DRG (2 d culture). Student’s unpaired t test; t = 9.060, p = 0.0008. n = 3 mice, or replicates per group.(I) LDH-release cytotoxicity assay of negative control or Raet1-selective siRNA knockdown embryonic DRG. Three replicate co-cultures for each siRNA group. Matched two-way ANOVA F(1,10) = 133.85, p < 0.0001).See also Figure S1. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/30712871), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Mouse NKp46/NCR1 Antibody by Immunohistochemistry Peripheral Nerve Injury Regulates Raet1 Expression and Injured Sensory Neurons Show Increased Neurite Fragmentation by Stimulated NK Cells. (H) Sciatic nerve tissue sections from adult male NKp46-YFP mice 7 days after L5 spinal nerve transection injury immunolabeled with anti-GFP (NKp46, green). Arrows indicate NK cells in sciatic nerve. Image collected and cropped by CiteAb from the following publication (https://linkinghub.elsevier.com/retrieve/pii/S0092867418316362), licensed under a CC-BY license. Not internally tested by R&D Systems.