Journal: The Journal of Clinical Investigation

Article Title: Loss of intestinal core 1-derived O -glycans causes spontaneous colitis in mice

doi: 10.1172/JCI45538

Figure Lengend Snippet: (A) Peripheral myeloid cells analyzed by flow cytometry (mean ± SD, n = 8 mice/group). Granulocytes are defined as Ly-6G+CD11b+. Ly-6G–CD11b+ monocytes were further analyzed for Ly-6C expression. *P < 0.02. (B) Histologic scores of H&E-stained colon sections of 2.5-week-old IEC C1galt1–/– mice treated with etanercept (TNF blocker; saline as control, mean ± SD, n = 5 mice/group) or blocking antibodies to mouse P- and E-selectin (anti-P/E, rat IgG; isotype IgG used as controls; mean ± SD, n = 6 mice/group). **P < 0.002, †P < 0.01. (C) Cryosections of IEC C1galt1–/– colons at different ages stained with mAbs to granulocytes (Ly-6G), macrophages (F4/80), and TNF. Insets (original magnification, ×400) highlight TNF+ granulocytes and macrophages. Scale bars: 50 μm. Data are representative of at least 3 experiments.

Article Snippet: Cryosections (20 μm thick) were incubated with rat mAb against murine Ly6G (5 μg/ml, clone 1A8; BD) or rat mAb against murine F4/80 (5 μg/ml, clone C1:A3-1; AbD Serotec) combined with rabbit anti–mouse TNF (polyclonal; BD) overnight at 4°C, developed with phycoerythrin-labeled goat anti–rabbit IgG (1:50; Jackson ImmunoResearch Laboratories Inc.) and Alexa Fluor 488–conjugated donkey anti–rat IgG (1:100; Invitrogen), and mounted with ProLong Gold mounting medium with DAPI (Invitrogen).

Techniques: Flow Cytometry, Expressing, Staining, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss of intestinal core 1-derived O -glycans causes spontaneous colitis in mice

doi: 10.1172/JCI45538

Figure Lengend Snippet: (A) Representative images of immunohistochemical staining (brown) with O-glycosylation–independent antibodies to different mucins in colon sections of WT and TM-IEC C1galt1–/– mice 5 days after TM. Scale bars: 100 μm. (B) Serum concentrations of FITC-dextran were measured 4 hours after oral administration to WT and TM-IEC C1galt1–/– mice at different time points after TM induction (mean ± SD, n = 6). *P < 0.008. (C) Relative amount of bacterial 16S rDNA detected by real-time PCR using two independent sets of 16S universal primers. Data are expressed as the fold difference between WT and TM-IEC C1galt1–/– mice (mean ± SD, n = 6). The average 16S rDNA value of WT mice was expressed as 1. **P < 0.02. (D) Colon sections of 7-week-old mice 10 days after TM induction were probed with an Alexa Fluor 555–conjugated general bacterial 16S rDNA probe for FISH (red) and costained with a FITC-labeled glycosylation-independent polyclonal antibody to Muc2 (green). Dashed lines mark epithelial surfaces. Scale bar: 20 μm. (E) Quantification of immunofluorescence images of 7-week-old WT and TM-IEC C1galt1–/– colon sections stained with mAbs to myeloid cells (Gr1) and TNF (mean ± SD, n = 5). †P < 0.001.

Article Snippet: Cryosections (20 μm thick) were incubated with rat mAb against murine Ly6G (5 μg/ml, clone 1A8; BD) or rat mAb against murine F4/80 (5 μg/ml, clone C1:A3-1; AbD Serotec) combined with rabbit anti–mouse TNF (polyclonal; BD) overnight at 4°C, developed with phycoerythrin-labeled goat anti–rabbit IgG (1:50; Jackson ImmunoResearch Laboratories Inc.) and Alexa Fluor 488–conjugated donkey anti–rat IgG (1:100; Invitrogen), and mounted with ProLong Gold mounting medium with DAPI (Invitrogen).

Techniques: Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction, Labeling, Immunofluorescence