primary antibodies against muc2 sc-7314  (Cell Signaling Technology Inc)

Journal: The Journal of Experimental Medicine

Article Title: PGD 2 and CRTH2 counteract Type 2 cytokine–elicited intestinal epithelial responses during helminth infection

doi: 10.1084/jem.20202178

Figure Lengend Snippet: Expression of Gpr44 in skin and lung and lineage-associated genes in sorted IECs. (a) Gpr44 expression in enriched CD45 + and CD45 − skin and lung fractions was determined using real-time PCR (MLN cells as positive control, calculated relative to Actβ ). (b–f) Enrichment of expression of characteristic epithelial cell markers Sis for enterocytes, Muc2 for goblet cells, Dclk1 for tuft cells, Lgr5 for ISCs, Chga for enteroendocrine cells, and Lyz1 for Paneth cells by sorted enterocyte-enriched fractions gated as live, EpCAM + UEA 1 − CD24 − (b), sorted goblet cell–enriched fractions gated as live, CD45 − EpCAM + UEA 1 + CD24 − (c), sorted tuft cell–enriched fractions gated as live, EpCAM + Siglec F + CD24 + (d), sorted ISC-enriched fractions gated as live, EpCAM + Lgr5GFP + (e), and sorted Paneth cell–enriched fractions gated as Defa6tdTomato + (f). Data are mean ± SEM. (a) n = 6; two independent experiments. (b–f) Five independent experiments from three or four pooled mice/experiment.

Article Snippet: Primary antibody against Muc2 (clone H-300; Santa Cruz Biotechnology) was diluted in immunofluorescence buffer (1% normal goat serum and 0.5% BSA), and staining was done overnight at 4°C or for 1 h at room temperature with slow agitation.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: PGD 2 and CRTH2 counteract Type 2 cytokine–elicited intestinal epithelial responses during helminth infection

doi: 10.1084/jem.20202178

Figure Lengend Snippet: CRTH2 limits murine small intestinal organoid budding and goblet cell accumulation. (a–c) Small intestinal organoids from littermate C57BL/6 WT and Gpr44 −/− mice were cultured and assessed directly after seeding. (a) Organoids were assessed for seeding efficiency at day 7 after seeding (calculated as number of organoids/well/input number of crypts, where the input number was equivalent for WT and Gpr44 −/− genotypes). (b and c) Representative light microscopy images on days 4, 5, 7, and 10 after seeding (b) were quantified for the number of buds/organoid in ∼50 organoids/genotype (c). Scale bar = 200 µm. (d and e) Representative images showing immunofluorescence staining for Muc2 (red) and DAPI (blue) in whole-mount organoids on day 10 after seeding (d) were quantified for Muc2 MFI (AU; e). Scale bar = 100 µm. (f and g) Representative plots (f) and frequencies of goblet cells (live, CD45 − EpCAM + UEA 1 + CD24 − ; g) assessed using flow cytometry in organoids at day 10 after seeding. Data are mean ± SEM. (a and g) Analyzed using an unpaired Student’s t test (only one value/experiment). ( c and e ) Analyzed using a linear mixed-effects model with pairwise comparison. (a–g) Three experiments with independent organoid lines for WT and Gpr44 −/− organoids. (c and e) 15–20 organoids/genotype/experiment analyzed. ***, P ≤ 0.001.

Article Snippet: Primary antibody against Muc2 (clone H-300; Santa Cruz Biotechnology) was diluted in immunofluorescence buffer (1% normal goat serum and 0.5% BSA), and staining was done overnight at 4°C or for 1 h at room temperature with slow agitation.

Techniques: Cell Culture, Light Microscopy, Immunofluorescence, Staining, Flow Cytometry, Comparison

Journal: The Journal of Experimental Medicine

Article Title: PGD 2 and CRTH2 counteract Type 2 cytokine–elicited intestinal epithelial responses during helminth infection

doi: 10.1084/jem.20202178

Figure Lengend Snippet: The PGD 2 -CRTH2 pathway limits the Type 2 cytokine–induced IEC functional program. (a–c) Littermate or cohoused C57BL/6 WT and Gpr44 −/− mice were infected with 500 N. brasiliensis (Nb) L3 larvae subcutaneously and treated i.p. with EdU on day 4 p.i. In naive (N) mice and mice on day 5 p.i., representative images showing immunofluorescent staining for EdU in histological sections of the small intestine (SI; a) were quantified for the ratio of the EdU (green) MFI to DAPI (blue) MFI (b) and the distance EdU + cells had traveled in the crypt/villus unit normalized to the total crypt/villus length (c). Scale bar = 100 µm. Small intestinal organoids from C57BL/6 WT mice were cultured for 2–3 d in media or Type 2 cytokines (250 µg/ml rmIL-4 and 250 µg/ml rm IL-13) with or without 20 µM PGD 2 and with or without 1 µM OC000459 (CRTH2 inhibitor). (d–f) Representative images showing immunofluorescence staining for EdU (green, upper row) or Muc2 (red, lower row) and DAPI (blue) in whole-mount organoids (d) were quantified for the EdU ratio to DAPI MFI (e) or Muc2 MFI (f). Scale bar = 100 µm. (g and h) Organoids were dissociated, sort-purified for live cells, and subjected to RNA-seq to identify DEGs that were down-regulated (downreg.; g) or up-regulated (upreg.; h) by culture with rmIL-4+rmIL-13 compared with vehicle alone and reversed when PGD 2 was added, in a CRTH2-dependent manner (top 50 displayed). (i and j) IPA was used to identify functional pathways that were enriched in these DEG sets, showing the top 20 ordered by P value of those with a z-score >2.0 (up-regulated) or less than −2.0 (down-regulated). (k) Il13ra1 expression (normalized counts) in organoids from RNA-seq data. Data are mean ± SEM. (b, c, e, and f) Analyzed using a linear mixed-effects model with pairwise comparison. (k) Analyzed using a one-way ANOVA. (a–c) N, n = 4 or 5; Nb, n = 4 or 5 (5–10 crypt/villus units were examined/mouse and those values were averaged); two independent experiments. (d–f) n = 5–10 organoids/condition/experiment; four independent experiments. (g–k) n = 3 replicates/condition; one experiment. Each n refers to number of mice/group in total across all experiments unless otherwise noted. *, P ≤ 0.05; **, P ≤ 0.01, ***, P ≤ 0.001. canc., cancer; dev., development; DSB, double-stranded break; ES, embryonic stem; infect., infection; mig., migration; move., movement; org., organization; prolif., proliferation; prog., progression.

Article Snippet: Primary antibody against Muc2 (clone H-300; Santa Cruz Biotechnology) was diluted in immunofluorescence buffer (1% normal goat serum and 0.5% BSA), and staining was done overnight at 4°C or for 1 h at room temperature with slow agitation.

Techniques: Functional Assay, Infection, Staining, Cell Culture, Immunofluorescence, Purification, RNA Sequencing, Expressing, Comparison, Migration

Journal: The Journal of Experimental Medicine

Article Title: PGD 2 and CRTH2 counteract Type 2 cytokine–elicited intestinal epithelial responses during helminth infection

doi: 10.1084/jem.20202178

Figure Lengend Snippet: Lgr5 expression in WT and Gpr44 −/− mice following N. brasiliensis infection and organoid TUNEL staining and gene expression in response to Type 2 cytokines with or without engagement of the PGD 2 -CRTH2 pathway. (a) C57BL/6 WT and Gpr44 −/− mice were infected with 500 N. brasiliensis (Nb) L3 larvae subcutaneously, and the expression of Lgr5 in small intestinal (SI) homogenates was measured by real-time PCR (relative to Actβ and normalized to WT naive [N]). Small intestinal organoids from C57BL/6 WT mice were cultured for 2 d in media or Type 2 cytokines (250 µg/ml rmIL-4 and 250 µg/ml rmIL-13) with or without 20 µM of PGD 2 and with or without 1 µM OC000459. (b) Real-time PCR was used to measure expression of Lgr5 calculated relative to Gapdh . (c) Representative images showing immunofluorescence staining for TUNEL in whole-mount organoids. Scale bar = 100 µm. (d) Real-time PCR was used to measure expression of Muc2 , Clca3 , Dclk1 , and Villin calculated relative to Gapdh . (e) Organoids were prestimulated (pre-stim) with Type 2 cytokines with or without 20 µM PGD 2 . Real-time PCR was used to measure expression of Muc2 and Clca3 , calculated relative to Gapdh . (f and g) Enrichment map of functional pathways associated with PGD 2 -responsive genes that were down-regulated (downreg.; f) or up-regulated (upreg.; g) by IL-4+IL-13 treatment of organoids. Related pathways were clustered and annotated in Cytoscape. Data are mean ± SEM. (a, b, d, and e) Analyzed using a linear mixed-effects model with pairwise comparison. (a) N, n = 10–12; Nb, n = 14–19; six independent experiments. (b) Three or four experiments. (c) Two experiments. (d and e) Three to six experiments. (f and g) n = 3 replicates/condition; one experiment. Each n refers to number of mice/group in total across all experiments unless otherwise noted. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Primary antibody against Muc2 (clone H-300; Santa Cruz Biotechnology) was diluted in immunofluorescence buffer (1% normal goat serum and 0.5% BSA), and staining was done overnight at 4°C or for 1 h at room temperature with slow agitation.

Techniques: Expressing, Infection, TUNEL Assay, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Immunofluorescence, Functional Assay, Comparison

Journal: PLoS ONE

Article Title: Spatial Localization and Binding of the Probiotic Lactobacillus farciminis to the Rat Intestinal Mucosa: Influence of Chronic Stress

doi: 10.1371/journal.pone.0136048

Figure Lengend Snippet: The example of vehicle-fed sham-stressed and stressed rats (control and WAS groups, respectively) is given. (A,C) sham-stressed rats; (B,D) stressed rats. FITC-labeled bacterial cells are seen in green, Muc2 is in red and cell nuclei are in blue (DAPI staining) (A,B: scale bar 20 μm; C,D: scale bar 5 μm). In the colon and independently of stress, L . farciminis bound to mucus, and notably to Muc2.

Article Snippet: In parallel, mucin immunohistochemical staining was realized by using polyclonal primary antibody against MUC2 (H300 sc-15334, Santa Cruz Biotechnology, diluted 1:100).

Techniques: Control, Labeling, Staining

Journal: PLoS ONE

Article Title: Spatial Localization and Binding of the Probiotic Lactobacillus farciminis to the Rat Intestinal Mucosa: Influence of Chronic Stress

doi: 10.1371/journal.pone.0136048

Figure Lengend Snippet: The example of vehicle-fed sham-stressed and stressed rats (control and WAS groups, respectively) is given. (A) sham-stressed rats; (B) stressed rats. FITC-labeled bacterial cells are seen in green, Muc2 is in red and cell nuclei are in blue (DAPI staining) (scale bar 5 μm). In the ileum and independently of stress, L . farciminis bound to mucus, and notably to Muc2.

Article Snippet: In parallel, mucin immunohistochemical staining was realized by using polyclonal primary antibody against MUC2 (H300 sc-15334, Santa Cruz Biotechnology, diluted 1:100).

Techniques: Control, Labeling, Staining

Journal: PLoS ONE

Article Title: Spatial Localization and Binding of the Probiotic Lactobacillus farciminis to the Rat Intestinal Mucosa: Influence of Chronic Stress

doi: 10.1371/journal.pone.0136048

Figure Lengend Snippet: The example of vehicle-fed sham-stressed and stressed rats (control and WAS groups, respectively) is given. Bacterial binding to BSA is shown as negative control. L . farciminis tightly bound to ileal and colonic Muc2 in stressed and sham-stressed animals.

Article Snippet: In parallel, mucin immunohistochemical staining was realized by using polyclonal primary antibody against MUC2 (H300 sc-15334, Santa Cruz Biotechnology, diluted 1:100).

Techniques: Control, Binding Assay, Negative Control