Mouse NKp46/NCR1 Antibody

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.