uea1 antibody  (Bioss)

Journal: The Journal of Experimental Medicine

Article Title: Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development

doi: 10.1084/jem.20102327

Figure Lengend Snippet: Expression of Xcr1 and Xcl1 in the thymus. (A–C) Quantitative RT-PCR analysis of Xcr1 expression in sorted thymus cell populations, including CD45 + thymocytes (CD45 + ), CD45 − thymic stromal cells (CD45 − ), CD45 − I-A + UEA1 + mTECs, CD11b + CD11c − macrophages (MΦ), and CD11c + tDCs (A); in sorted tDC subpopulations, including CD11c + total tDCs, I-A high CD11c high CD11b − lymphoid tDCs (lyDC), I-A high CD11c high CD11b + myeloid tDCs (mDC), and I-A medium CD11c medium B220 + plasmacytoid tDCs (pDC) (B); and in CD11c + DCs from the thymus (Thy), the spleen (Spl), the subcutaneous lymph nodes (subLN), and the mesenteric lymph nodes (mesLN; C). The amounts of Xcr1 were normalized to the amount of Hprt , and those in CD45 + thymocytes (A) and total tDCs (B and C) were arbitrarily set to 1. (D and E) Quantitative RT-PCR analysis of Xcl1 expression in thymus cell populations, including CD45 + thymocytes (CD45 + ), CD11c + DCs, CD45 − I-A − MTS15 + fibroblasts (Fib), CD45 − I-A + total TECs (TEC), CD45 − I-A + Ly51 + cTECs (cTEC), and CD45 − I-A + UEA1 + mTECs (mTEC); and in CD45 − I-A low UEA1 + (MHC II lo ) and CD45 − I-A high UEA1 + (MHC II hi ) mTEC subpopulations. The amounts of Xcl1 were normalized to the amount of Hprt , and those in CD45 + thymocytes (D) and CD45 − I-A low UEA1 + mTECs (E) were arbitrarily set to 1. Bar graphs show means and standard errors of at least three independent measurements. (F) Quantitative RT-PCR analysis of Xcl1 expression in CD45 − I-A + UEA1 + mTECs and anti-CD3–stimulated CD8 + T cells. Means (bars) and individual measurements (circles) are shown ( n = 3).

Article Snippet: Frozen thymus tissues embedded in OCT compound (Sakura) were sliced into 5-µm-thick sections, fixed with acetone, and stained with the following antibodies: mTEC-specific monoclonal antibody ER-TR5 (a gift from W. van Ewijk, Erasmus University, Rotterdam, Netherlands) followed by Alexa Fluor 633–conjugated anti–rat IgG antibody (Invitrogen); biotinylated UEA1 (Vector Laboratories) followed by Alexa Fluor 633–conjugated streptavidin (Invitrogen); FITC-conjugated anti-Foxp3 monoclonal antibody (eBioscience); biotinylated anti-CD11c or anti-CD11b monoclonal antibody (eBioscience) followed by Alexa Fluor 488–conjugated or Alexa Fluor 546–conjugated streptavidin (Invitrogen); and anti-Aire antibody (Santa Cruz Biotechnology, Inc.) followed by FITC-conjugated anti–rabbit IgG antibody (Invitrogen).

Techniques: Expressing, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development

doi: 10.1084/jem.20102327

Figure Lengend Snippet: DCs and T reg cells in the thymus of Aire -deficient mice. (A) Quantitative RT-PCR analysis of Xcl1 , Ccl19 , Ccl21 , and Ccl25 expression in sorted CD45 − EpCAM + UEA1 + mTECs from WT and Aire -deficient mice. The amounts of the transcripts were normalized to the amount of housekeeping Hprt , and those in WT mTECs were arbitrarily set to 1. Bar graphs show means and standard errors of at least three independent measurements. (B) Immunofluorescence analysis of thymus sections for CD11c (green) and mTECs (ER-TR5; red). Representative images from two independent experiments are shown. Lines indicate borders among the indicated regions identified as in Fig. S2 A . (C) Numbers of CD11c + cells per unit area (1 mm 2 ) of the indicated regions were measured. Means and standard errors of cell numbers are shown. (D) Means and standard errors ( n = 3–4) of the absolute numbers of indicated DC subpopulations in the thymus of indicated mouse strains are shown. (E) Immunofluorescence analysis of thymus sections from indicated mice for Foxp3. Representative images from two independent experiments are shown. (F) Numbers of Foxp3 + cells per unit area (1 mm 2 ) of the indicated areas were measured. Means and standard errors of cell numbers from three independent measurements are shown. (G) Means and standard errors ( n = 3) of the absolute numbers of CD4 + CD8 − CD25 + Foxp3 + cells in the thymus of indicated mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, not significant (P > 0.05).

Article Snippet: Frozen thymus tissues embedded in OCT compound (Sakura) were sliced into 5-µm-thick sections, fixed with acetone, and stained with the following antibodies: mTEC-specific monoclonal antibody ER-TR5 (a gift from W. van Ewijk, Erasmus University, Rotterdam, Netherlands) followed by Alexa Fluor 633–conjugated anti–rat IgG antibody (Invitrogen); biotinylated UEA1 (Vector Laboratories) followed by Alexa Fluor 633–conjugated streptavidin (Invitrogen); FITC-conjugated anti-Foxp3 monoclonal antibody (eBioscience); biotinylated anti-CD11c or anti-CD11b monoclonal antibody (eBioscience) followed by Alexa Fluor 488–conjugated or Alexa Fluor 546–conjugated streptavidin (Invitrogen); and anti-Aire antibody (Santa Cruz Biotechnology, Inc.) followed by FITC-conjugated anti–rabbit IgG antibody (Invitrogen).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence