anti uea1 biotin  (Vector Laboratories)

Journal: Nature Communications

Article Title: The alarmin IL33 orchestrates type 2 immune-mediated control of thymus regeneration

doi: 10.1038/s41467-023-43072-x

Figure Lengend Snippet: a Representative FACs plots illustrating the proportion of IL33 cit expressing CD45 + thymic cells and total CD45 - thymic stromal cells (blue histogram). Red histograms represent florescence levels in control WT mice. b Quantitation of IL33 cit expression in defined thymic stromal populations; cTEC (CD45 - EpCAM-1 − UEA1 − Ly51 + ), mTEC (CD45 - EpCAM-1 − UEA1 + Ly51 − ), mTEC hi (CD45 - EpCAM-1 − UEA1 + Ly51 − MHCII + CD80 + ), mTEC lo (CD45 - EpCAM-1 - UEA1 + Ly51 − MHCII - CD80 − ), Endothelium (TER119 − CD45 − EpCAM-1 − CD31 + ), Mesenchyme (TER119 − CD45 − EpCAM-1 − CD31 − Integrinα7 − ), Pericytes (TER119 − CD45 − EpCAM-1 − CD31 − Integrinα7 + ), (where n = 7 animals for TEC subsets and n = 5 animals for all other subsets, obtained from 2 independent experiments). c Representative FACs plots showing Sca-1 expression in thymic mesenchyme, and IL33 cit expression within Sca-1 + and Sca-1 − subsets. Blue histograms are from IL33 cit mice, red histograms from control WT mice. d Total thymus cellularity, Sca-1 + mesenchyme numbers/proportions in WT mice following SLI exposure at indicated time points ( n = 6 animals obtained from 2 independent experiments) with statistical analysis from a one-way ANOVA comparing means of each group to D0 with Dunnett’s multiple comparisons. e Comparative analysis of TEC (blue line) and Sca-1 + mesenchyme (red line) recovery at indicated time points in WT mice ( n = 6 animals obtained from 2 independent experiments). f FACs plots showing IL4Rα expression in Sca-1 + mesenchyme from WT (blue histogram) mice, where against Il4ra −/− mice (red histogram) were used for control staining levels. g Quantitation of Sca1 + mesenchyme in WT and Il4ra −/− mice at steady state (D0) (WT n = 5 and Il4ra −/− n = 6 animals obtained from 2 independent experiments), from a two-tailed unpaired Student’s t test. h Injection and irradiation regime of 5μg recombinant IL4-complexes/PBS into WT mice. Analysis of Sca-1 + mesenchyme was performed at day 7 and day 14 after injection regime, n = 6 minimum. i Numbers of Sca-1 + thymic mesenchyme cells at day 7 and day 14 post-SLI following PBS (D7 n = 6 and D14 n = 7 animals) or IL4c (D7 n = 7 and D14 n = 7 animals) injection, obtained from 2 independent experiments. Statistics from a two-tailed unpaired Student’s t test. All analysis was carried out across at least two independent experiments. All error bars show mean ± SEM.

Article Snippet: For TEC, mesenchyme, and endothelial analysis , , thymus tissues were digested with collagenase dispase (2.5 mg/ml) (Roche) and DNase I (100 mg/ml) (Roche) before being stained with the following antibodies/reagents: anti-EpCAM1 clone G8.8, Brilliant Violet 711 (1:400), PerCP-eFluor 710 (1:2000), eBioscience, UEA1 Biotin (1:10000) (Vector Labs) detected using streptavidin PE Cy7 (1:1500), eBioscience, anti-Ly51 clone BP-1, PerCP-eFluor 710 (1:600), BD Pharmingen, anti-MHCII clone M5/114.15.2, Alexa Fluor 700 (1:1200), eBioscience, anti-CD80 clone 16-10A1, Brilliant Violet 605 (1:400), BioLegend, anti-CD31 clone 390, PE Cy7 (1:800), Invitrogen, anti-Sca-1 clone D7, PE (1:2000), FITC (1:1000), eBioscience, anti-IL4Ra clone I015F8, PE (1:50), BioLegend, anti-Integrinα7 clone 334908, Alexa Fluor 700 (1:100), R&D, and Zombie Aqua Fixable Viability Kit, Brilliant Violet 510 (1:1000), BioLegend.

Techniques: Expressing, Quantitation Assay, Staining, Two Tailed Test, Injection, Irradiation, Recombinant