Journal: Acta biomaterialia

Article Title: Use of hydrogel scaffolds to develop an in vitro 3D culture model of human intestinal epithelium.

doi: 10.1016/j.actbio.2017.08.035

Figure Lengend Snippet: Fig. 7. Immunohistochemistry staining (brown) of MUC2 and MUC5AC; A: monolayer and IgG as a negative control. HT29-MTX cells layered on or suspended within B: alginate, C: L-pNIPAM, and D: L-pNIPAM-co-DMAc hydrogels under static or dynamic culture conditions at a cell density of 2 106 cells/ml following 21 days. Cell nuclei were stained with haematoxylin (blue). Yellow arrows indicate positively stained cells. Scale bar = 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Immunohistochemistrywas performed to investigate: brush border differentiation using CD10 antibody (1:100 rabbit polyclonal, enzyme antigen retrieval) (Abcam, Cambridge, UK); Zonulin 1 (ZO-1) protein expression which is a tight junction protein expressed by enterocytes using ZO-1 antibody (1:50, enzyme antigen retrieval) (Abcam, Cambridge, UK); enterocyte differentiation markers: alkaline phosphatase (ALP) antibody (1:200 rabbit polyclonal, heat antigen retrieval) (Abcam, Cambridge, UK), dipeptidyl peptidase IV (DPP IV) antibody (1:50mousemonoclonal, enzyme antigen retrieval) (Abcam, Cambridge, UK); and sucraseisomaltase antibody (SI) (1:50, mouse monoclonal antibody, heat antigen retrieval) (Santa Cruz, Heidelberg, Germany); HT29-MTX differentiation was assessed using MUC2 antibody (1:100 rabbit polyclonal, heat antigen retrieval) (Santa Cruz, Heidelberg, Germany) and MUC5AC antibody (1:200, mouse monoclonal antibody, heat antigen retrieval) (Abcam, Cambridge, UK).

Techniques: Immunohistochemistry, Staining, Negative Control

Journal: Nature Communications

Article Title: FAM3D is essential for colon homeostasis and host defense against inflammation associated carcinogenesis

doi: 10.1038/s41467-020-19691-z

Figure Lengend Snippet: a Heatmaps of differentially expressed genes enriched in host defense responses. b mRNA expression levels of antibacterial peptides in the colons of WT or Fam3D −/− mice measured by real-time PCR. n = 8. c Reg3γ protein levels measured by immunofluorescent staining. n = 4 for each group. d GSEA analysis on Fam3D −/− vs WT EBSeq log 2 FC expression data of colonic epithelial samples. e Immunofluorescent staining for Fam3D and MUC2 in normal colon tissue. Data is representative of one experiment repeated three independent times. f Double staining of Acian blue and PAS. Yellow arrow: Acian blue positive materials; orange arrow: PAS single positive cells; orange star: dilated goblet cells, goblet cells enumerated in each crypt (10–20 crypts from each colon) and pixels of Alcian blue staining areas. Alcian blue staining areas and fluorescence intensity quantified by Image J based on six distal colon sections of four independent mice from each group. g Staining of high iron diamine/Alcian blue, dark brown, and black were sulfomucins, whereas blue staining indicates sialomucins, sulfomucin positive cells enumerated in each crypt (10–20 crypts from each colon) and the ratio of sialomucins to sulfomucins is calculated. Data is presented as the mean ± SEM. Scale bar = 50 μm. h Representative Alcian blue staining of Carnoy’s-fixed colonic sections. The thickness of the inner mucus layer (light blue band between luminal content and the mucosa) was quantified (5–10 fields from each colon) by Image J. n = 8. i Representative dual staining for UEA-I (immunofluorescence, green) and bacteria (fluorescence in situ hybridization, red), the latter using the universal EUB338 probe, on Carnoy’s-fixed colonic sections. n = 8. j PAS/Alcian blue staining of the colons from WT and Fam3D −/− mice with different ages. n = 4. Data is representative of one experiment repeated three independent times. k Quantitation of crypt length of WT and Fam3D −/− mice at different ages. Data is presented as the mean ± SEM. Statistical significance was determined by unpaired, two-tailed Student’s t test.

Article Snippet: Rat anti-EpCAM antibody (sc-53532), mouse anti-β-catenin antibody (sc-7963), rabbit anti-mouse Mucin 2 (MUC2) antibody (H300) were purchased from Santa Cruz (Dallas, TX).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Staining, Double Staining, Fluorescence, Immunofluorescence, Bacteria, In Situ Hybridization, Quantitation Assay, Two Tailed Test

Journal: Mucosal immunology

Article Title: Hypoxia-inducible factor 1 in dendritic cells is crucial for the activation of protective regulatory T cells in murine colitis.

doi: 10.1038/mi.2015.67

Figure Lengend Snippet: Figure 6 Dendritic cell-specific knockout of hypoxia-inducible factor-1a (HIF-1a) stimulates mucosal epithelium to increased production of mucins in dextran sodium sulfate (DSS) colitis. Colonic mRNA expression of (a) MUC1, (b) MUC2, and (c) MUC3 was significantly higher in DSS-treated CD11cCre/HIF-1a þ f/ þ f mice than in HIF-1a þ f/ þ f DSS-treated mice or in mice not treated with DSS. (d) Sections (4 mm) of colon tissue were stained with MUC2 (red) and 4’,6-diamidino-2-phenylindole (DAPI; blue). After treatment with DSS, the amount of colonic MUC2 protein was higher in CD11cCre/HIF- 1a þ f/ þ f mice than in HIF-1a þ f/ þ f mice (original magnification 200; scale bar ¼ 50 mm). N ¼ 5–7 per group. *Po0.05; **Po0.01; ****Po0.0001. C, control; NS, not significant.

Article Snippet: MUC2 was detected with a rabbit anti-mouse MUC2 antibody (Santa Cruz Biotechnology).

Techniques: Knock-Out, Expressing, Staining, Control

Journal: Gut Pathogens

Article Title: Effect of crude polysaccharide from seaweed, Dictyopteris divaricata (CDDP) on gut microbiota restoration and anti-diabetic activity in streptozotocin (STZ)-induced T1DM mice

doi: 10.1186/s13099-022-00512-1

Figure Lengend Snippet: Crude polysaccharide (CDDP) enhanced the expression of: A Mucin-2 (MUC-2) and B Zonula occludens-1 (ZO-1) in STZ-induced T1DM mice colon by immunohistochemistry technique after 4-weeks treatment. Inflammatory cells (demonstrated by red arrow), goblet cells (demonstrated by yellow arrow). Mucin expression (demonstrated by green arrow) and Zonula occludens-1 expression (demonstrated by orange arrow) were shown. The picture is the representative of colon of six different treatment groups. Original magnification: 20x, scale bar: 100 µm

Article Snippet: The primary antibodies [goat antirabbit: β-actin, Mucin-2 (MUC-2), Claudin-1, Occludin, Zonula occludens-1 (ZO-1) and insulin receptor substrate-1 (IRS-1)], secondary antibodies and the radioimmunoprecipitation assay (RIPA) buffer were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Immunohistochemistry

Journal: Gut Pathogens

Article Title: Effect of crude polysaccharide from seaweed, Dictyopteris divaricata (CDDP) on gut microbiota restoration and anti-diabetic activity in streptozotocin (STZ)-induced T1DM mice

doi: 10.1186/s13099-022-00512-1

Figure Lengend Snippet: Effect of CDDP treatment on tight junction proteins (TJs) expression in the colon epithelium. A The relative expression of Occludin and Claudin-1 in STZ-induced T1DM mice were analyzed by western blotting technique after 4-weeks treatment using β-actin as an internal control. Western blots are the representative of three experiments. B Bar graph represent the intensity of relative protein band against β-actin as an internal control, quantified by NIH image J software. Data obtained from three experiments are presented as Mean ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.001, model group compared with control group and CDDP and metformin treatment groups compared with model group

Article Snippet: The primary antibodies [goat antirabbit: β-actin, Mucin-2 (MUC-2), Claudin-1, Occludin, Zonula occludens-1 (ZO-1) and insulin receptor substrate-1 (IRS-1)], secondary antibodies and the radioimmunoprecipitation assay (RIPA) buffer were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Western Blot, Control, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sialic acid plays a pivotal role in licensing Citrobacter rodentium's transition from the intestinal lumen to a mucosal adherent niche.

doi: 10.1073/pnas.2301115120

Figure Lengend Snippet: Fig. 1. Citrobacter rodentium resides within the colonic mucus and catabolizes sialic acid. (A) C. rodentium localizes to the mucus layer. Representative immunofluorescence staining of mouse colonic tissue infected with C. rodentium. A colon cross-section (green panel) was stained with DAPI to detect DNA (blue), anti-C. rodentium (red) to visualize C. rodentium, and anti-Muc2 to visualize mucus (green). The gray panel is the enlarged view of the boxed region within the cross-section, original magnification = 200×. The orange panel is a magnified image indicating a subpopulation of C. rodentium localized to the inner mucus and traversing the mucus (arrowheads), with a separate image showing C. rodentium staining independently (red channel), original magnification = 630×. (Scale bar, 15 μm.) (B) C. rodentium uses sialic acid as a sole carbon source for growth. C. rodentium growth was measured by optical density (OD600) at 20-min intervals over 24 h at 37 °C in M9 minimal medium supplemented with 0.2% N-acetylneuraminic acid (sialic acid) or purified mucins. Data are presented as averages of cell growth (n = 9) from three independent experiments.

Article Snippet: For visualizing C. rodentium localization in the mucus, methacarn- fixed mouse distal colons were stained with the following primary antibodies—rat anti- C. rodentium Tir (gift from W. Deng), rabbit anti- Muc2 (Boster), and rabbit anti- Muc2 (Novus), which were then probed with Alexa Fluor 488- conjugated donkey anti- rabbit IgG (Life Technologies) and Alexa Fluor 568–conjugated donkey anti- rat IgG (Life Technologies).

Techniques: Immunofluorescence, Staining, Infection, Purification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Sialic acid plays a pivotal role in licensing Citrobacter rodentium's transition from the intestinal lumen to a mucosal adherent niche.

doi: 10.1073/pnas.2301115120

Figure Lengend Snippet: Fig. 2. Sialic acid in the colon is mainly derived from mucus produced by goblet cells and widely expressed before and during C. rodentium infection. (A) Representative immunofluorescence staining of sialic acid on murine colonic sections with and without C. rodentium infection. Sections were stained with DAPI to detect DNA (blue) and SNA lectin (α2,6-sialic acid binding) to visualize sialic acid. Dotted lines indicate the apical side of the epithelium. Original magnification = 200×. (Scale bar, 50 µm.) (B) Degree of sialylation on Muc2 O-glycans of colonic mucus with (n = 4) and without (n = 4) infection with C. rodentium for 6 d. Released O-glycans from distal intestine were analyzed on PGC-LC-MS/MS. (C) Levels of free sialic acid in fecal contents of mice without (n = 6) and with (n = 6) C. rodentium infection for 6 d. All data are shown as mean ± SEM. Statistical significance calculated by the Mann–Whitney U test (B and C).

Article Snippet: For visualizing C. rodentium localization in the mucus, methacarn- fixed mouse distal colons were stained with the following primary antibodies—rat anti- C. rodentium Tir (gift from W. Deng), rabbit anti- Muc2 (Boster), and rabbit anti- Muc2 (Novus), which were then probed with Alexa Fluor 488- conjugated donkey anti- rabbit IgG (Life Technologies) and Alexa Fluor 568–conjugated donkey anti- rat IgG (Life Technologies).

Techniques: Derivative Assay, Produced, Infection, Immunofluorescence, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY