polyclonal goat anti mouse il25 antibody  (R&D Systems)

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, Co-localization of DCLK1 and RFP (IL25) in Flare25 thymus. Confocal maximal projection. Scale, 5 μm. n = 3 mice, 2 independent experiments. b, Differential expression of 20 tuft markers comparing sorted enterocytes and SI tuft cells (n = 3 mice) and thymic tuft cells (n = 4 mice) from Flare25 mice. Log2 fold change relative to mean expression. c, Expression (normalized reads from b ) of MHCII genes in SI and thymic tuft cells (mean +/− SD). * FDR < 0.00001. Lamp , FDR = 1. d, MHCII (I-A b ) surface staining on SI and thymic tuft cells. n = 3 mice; 2 independent experiments. e, Heatmap of select Tas2r genes comparing expression (normalized reads from b ) between thymic and SI tuft cells. f, Single cell RNA sequencing of RFP + (IL25 + ) thymic tuft cells. Heatmap shows selected tuft and Tas2r gene expression. Columns are single cells (n = 195), values are mean-centered log-normalized.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Quantitative Proteomics, Expressing, Staining, RNA Sequencing, Gene Expression

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, DCLK1 bright mTEC counts. n = 5 mice per genotype; 3 independent experiments. b, mTECs from iALT.Flare25 double-reporter mice after 10-day tamoxifen treatment. Right, MHCII (I-A b ) surface expression within each gated population. n = 5 mice; 2 independent experiments. c, Single cell RNA sequencing of RFP + (IL25) Aire −/− thymic tuft cells. Heatmap shows selected tuft and Tas2r gene expression. Columns are single cells (n = 95), values are mean-centered log-normalized. d, PCA of single cell RNA sequencing data from RFP + tuft cells. Right, Gnat3 expression overlaid. e, DCLK1 staining with signal converted to Imaris surfaces; volumes > 5x mean single-cell volume (1×10 4 μm 3 ) are pseudocolored according to size. Right, pooled data. n = 3 mice per genotype. f, KRT10 surface area in thymic medulla (per unit area). Three regions analyzed per thymus. n = 4 mice per genotype; 2 independent experiments. g, h, Analysis of DCLK1 bright cells in Hipk2 fl/fl controls or FoxN1-Cre.Hipk2 fl/fl thymus. g, Confocal imaging of IF staining. h, Frequencies of DCLK1 hi MHCII lo or DCLK1 + MHCII hi mTECs from flow analysis. n = 6 mice; 3 independent experiments. e, g, Scale, 100 μm. a, e, f, h, Mean +/− SD; unpaired, parametric, 2-tailed Student’s t test.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Expressing, RNA Sequencing, Gene Expression, Staining, Imaging

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, DT-treated Balb/cByJ. KN2 x C57BL/6. Aire-DTR F1 thymus (n = 10 mice) or non-Tg controls (n = 22 mice) gated on TCRβ int CD1d + iNKTs. Right, counts of IL4-producing (hCD2 + ) NKT2s (PLZF + ). b, DCLK1 intracellular staining in mTECs. n = 5 mice; 3 independent experiments. c, Thymic NKT2 (TCRβ int CD1d + PLZF + RORγt − ) cell counts in B6 (n = 15 mice), Pou2f3 −/− (n = 19 mice) and Trpm5 −/− (n = 10 mice). d, Thymic EOMES + TCRβ + CD8 + SP cell counts in B6 (n = 10 mice), Pou2f3 −/− (n = 11 mice) and Trpm5 −/− (n = 9 mice). e, Splenic total iNKT and NKT2 cell counts in B6 (n = 5 mice) and Pou2f3 −/− (n = 4 mice). f, Serum anti-IL25 autoantibody indices (AI) in nude mice transplanted with C57BL/6 (n = 3 mice) or Pou2f3 −/− (n = 12 mice) neonatal thymus and immunized with IL25 protein. IMM = immunized. Dashed line = average of C57BL/6 AI values plus 3 standard deiviations. of a, c–f, Mean +/− SD. a, e, Unpaired, parametric, 2-tailed Student’s t test. c, d, One-way, non-parametic ANOVA (Kruskal-Wallis test). a , c , d, Pooled from 3 independent experiments.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Staining

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, DCLK1 and RFP (IL25) in C57BL/6 control and Flare25 thymus. Confocal maximal projection. Scale, 5 μm. n = 3 mice, 2 independent experiments. b, RFP (IL25) in C57BL/6 control and Flare25 mTECs (CD11c − CD45 − EPCAM + ). n = 5 mice; 3 independent experiments. c, Gating strategy for FACS sorting of CD11c − CD45 − Epcam + Ly51 − mTECs from Flare25 reporter mice for qPCR analysis of mTEC hi (RFP − MHCII hi ), mTEC lo (RFP + MHCII lo ), and thymic tuft (RFP + ) populations. d, qPCR analysis of expression of indicated genes of interest on populations sorted in ( c ) normalized to mTEC hi (mean +/− SD). n = 3 mice. Two-way non-parametric ANOVA with multiple comparisons. e, Representative confocal maximum projection (10 μm) stained for KRT8/KRT18 (red) and DCLK1 (green). Scale bars, 50 μm. n = 3 mice, 2 independent experiments. f–i, Expression levels (normalized reads from ) from bulk RNA sequencing of SI (n = 3 mice) and thymic tuft (n = 4 mice) cells. f, Major MHCI genes and B2m. FDR > 0.1. g, Major MHCII genes and CD74 in SI tuft cells. h, Minor MHCII genes in thymic tuft cells. i, Tas2r family members in thymic tuft cells. g–i, Mean +/− SD. g, i, Red line corresponds to a cutoff of 5 reads/million; * mean and SD fall above this cutoff.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Control, Expressing, Staining, RNA Sequencing