biotinylated anti dll4  (R&D Systems)

Journal: The Journal of Experimental Medicine

Article Title: Delta-like 4 is indispensable in thymic environment specific for T cell development

doi: 10.1084/jem.20080134

Figure Lengend Snippet: Dll4 was expressed on thymic epithelium. The expression of Dll4 on the epithelium was detected in thymus (A and B, Thymus), but not gut (C, Gut) or salivary gland (C, Sal. G.) by immunofluorescence histochemistry. Staining was done with anti–pan-cytokeratin (CK; A, green), anti-K5 (K5; B, green), anti-K8 (K8; B, red), anti-Dll4 (Dll4; A, red; B, blue), or control IgG (IgG; A, red; B, blue) antibodies and analyzed by confocal laser microscopy. (A and B, right) High-magnification images of the red square in the middle images. (B, left) Hemotoxylin and eosin staining of serial section. Bars: (A, left and middle) 50 μm; (A, right) 10 μm; (B, left) 200 μm; (B, right) 10 μm; (C) 20 μm.

Article Snippet: Tissue sections of thymus were stained with FITC-anti-pan-cytokeratin (Sigma-Aldrich), biotinylated goat anti-Dll4 (R&D Systems) antibodies, biotinylated goat IgG, nonlabeled anti-K5 (Covance), anti-K8 (TROMA-I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or anti-CD31 (eBioscience) antibodies.

Techniques: Expressing, Immunofluorescence, Staining, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: Delta-like 4 is indispensable in thymic environment specific for T cell development

doi: 10.1084/jem.20080134

Figure Lengend Snippet: Specific abrogation of Dll4 on thymic epithelium. (A) Targeted insertion of loxP sequences flanking a part of exon 1 and whole exons 2 and 3 of the Dll4 gene. Numbers indicate exon number. 1*, first exon modified with loxP sequence; 1**, part of exon 1 after gene deletion; ATG, translational initiation codon of Dll4 ; triangles, loxP sequences; open boxes, Dll4 exons; B, BglII; C, ClaI; RI, EcoRI; Xb, XbaI; Xh, XhoI. (B) Cre activity was targeted to thymic epithelia using mice, designated FoxN1-Cre, in which this recombinase is under the transcriptional control of the FoxN1 locus. The timing and specificity of Cre-mediated recombination was visualized by the expression of enhanced GFP (eGFP) in thymus tissue sections of E12.5 (left) and 6-wk-old (right) crosses of FoxN1-Cre mice with the transgenic Z/EG reporter mice. The thymus anlage in the left image is outlined with a dashed line. C, cortex; M, medulla. (C) The expression of Dll4 on thymic epithelial, but not endothelial, cells was abrogated in Dll4 lox/lox FoxN1-Cre (FoxN1Cre:Dll4-floxed) mice. Epithelial and endothelial cells in the thymus were identified by the expression of cytokeratin (CK, green, left and middle) and CD31 (red, right), respectively, with Dll4 (red, middle; green, right). Bars: (B and C) 50 μm.

Article Snippet: Tissue sections of thymus were stained with FITC-anti-pan-cytokeratin (Sigma-Aldrich), biotinylated goat anti-Dll4 (R&D Systems) antibodies, biotinylated goat IgG, nonlabeled anti-K5 (Covance), anti-K8 (TROMA-I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or anti-CD31 (eBioscience) antibodies.

Techniques: Modification, Sequencing, Activity Assay, Expressing, Transgenic Assay

Journal: The Journal of Experimental Medicine

Article Title: Delta-like 4 is indispensable in thymic environment specific for T cell development

doi: 10.1084/jem.20080134

Figure Lengend Snippet: Notch signaling was decreased in the thymus with Dll4-null epithelial cells. (A) The cleaved Notch1 fragment was found in fetal thymocytes of E15.5 Dll4 lox/lox (Dll4-floxed) embryos. The cells with cleaved Notch1 are indicated with arrowheads. Bar, 10 μm. (B) Frequencies of cells with cleaved Notch1 (ICN1 + ; mean percentage from five fields in a slide with >100 cells from each embryo ± SD) were counted in E15.5 WT ( n = 3), Dll4 lox/lox FoxN1Cre (FoxN1Cre:Dll4-floxed, n = 6), or Dll4 lox/lox (Dll4-floxed, n = 6) mice. The cultured DN cells were prepared after the FTOC of E14.5 WT fetal thymus for 4 d with γ-secretase inhibitor (GSI, n = 3) or without γ-secretase inhibitor (DMSO, n = 3). Asterisk indicates unpaired Student's t test; P < 0.001.

Article Snippet: Tissue sections of thymus were stained with FITC-anti-pan-cytokeratin (Sigma-Aldrich), biotinylated goat anti-Dll4 (R&D Systems) antibodies, biotinylated goat IgG, nonlabeled anti-K5 (Covance), anti-K8 (TROMA-I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or anti-CD31 (eBioscience) antibodies.

Techniques: Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Delta-like 4 is indispensable in thymic environment specific for T cell development

doi: 10.1084/jem.20080134

Figure Lengend Snippet: No T cells, but substantial B cells, appear in the thymus with Dll4-null epithelium. (A) Thymic cellularity (mean ± SD) of fetus (E15.5; left, n = 3; right, n = 5), neonate (N.B.; left, n = 5; right, n = 3), 4-wk-old (4W, n = 4), or 8-wk-old (8W, n = 3) Dll4 lox/lox FoxN1-Cre (FoxN1Cre:Dll4-floxed, shaded columns) mice compared with Dll4 lox/lox (Dll4-floxed, open columns) mice. Asterisks indicate unpaired Student's t test; P < 0.001. (B) Flow cytometry of thymocytes from young adult (4W) Dll4 lox/lox mice with (bottom, FoxN1Cre:Dll4-floxed) or without (top, Dll4-floxed) FoxN1-Cre transgene. Thymocytes were stained with monoclonal antibodies to surface molecules as shown. Numbers in the profiles indicate the relative percentages, in CD4 − CD8 − cells (right, CD4+CD8 vs. Thy1.2), for each corresponding quadrant or fraction. (C) The ETP population was not observed in Dll4 lox/lox FoxN1-Cre (4W) mice. Thymocytes from Dll4 lox/lox (top) or Dll4 lox/lox FoxN1-Cre mice (bottom) were stained for lineage markers (Lin.: CD4, CD8, CD3, B220, CD19, Gr1, CD11b, TER119), CD44, CD25, CD24, and c-kit. Profiles are shown with the gate (Lin. − , middle; Lin. − CD44 + CD25 − as DN1, right). Numbers on the plots represent the frequency of cells lying in the indicated regions within the gate. The ETP population is identified as Lin. − CD44 + CD25 − CD24 − or low c-kit + cells (DN1a+b). DN1c and DN1–3 populations are also shown in the plots. Data are representative of three experiments.

Article Snippet: Tissue sections of thymus were stained with FITC-anti-pan-cytokeratin (Sigma-Aldrich), biotinylated goat anti-Dll4 (R&D Systems) antibodies, biotinylated goat IgG, nonlabeled anti-K5 (Covance), anti-K8 (TROMA-I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or anti-CD31 (eBioscience) antibodies.

Techniques: Flow Cytometry, Staining

Journal: The Journal of Experimental Medicine

Article Title: Delta-like 4 is indispensable in thymic environment specific for T cell development

doi: 10.1084/jem.20080134

Figure Lengend Snippet: Hematopoietic progenitors with active form of Notch1 give rise to T cell lineage in Dll4-deficient thymus. Hematopoietic progenitors (Lin. − c-kit + cells) derived from E15.5 fetal liver were infected with the retrovirus encoding the intracellular region of Notch1 (ICN1) or empty vector (Mock). Infected cells were monitored by expression of GFP. These cells were cultured into deoxyguanosine-treated thymic lobes of Dll4 lox/lox FoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4 lox/lox (Dll4-floxed) fetuses for 13 d as FTOC and analyzed for the expression of CD4, CD8, CD25, and CD19 (A). Alternatively, these cells were injected intravenously into irradiated Dll4 lox/lox FoxN1-Cre (FoxN1Cre:Dll4-floxed) or Dll4 lox/lox (Dll4-floxed) neonate mice. After 3 wk, thymocytes were analyzed for expression of CD4, CD8, and CD19 (B). Profiles are shown with the gate (GFP + or GFP + CD4 − CD8 − , DN-gated in A). Numbers in the profiles indicate the relative percentages for each corresponding quadrant.

Article Snippet: Tissue sections of thymus were stained with FITC-anti-pan-cytokeratin (Sigma-Aldrich), biotinylated goat anti-Dll4 (R&D Systems) antibodies, biotinylated goat IgG, nonlabeled anti-K5 (Covance), anti-K8 (TROMA-I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), or anti-CD31 (eBioscience) antibodies.

Techniques: Derivative Assay, Infection, Plasmid Preparation, Expressing, Cell Culture, Injection, Irradiation