Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Primers for quantitative RT-PCR

Article Snippet: After blocking with PBS containing 3% BSA, 0.5 μg/ml rabbit anti-mouse MUC2 polyclonal antibody (H-300; Santa Cruz Biotechnology) or IgG from rabbit serum (Sigma) was added to the wells and incubated overnight.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Autophagy Protects against Colitis by the Maintenance of Normal Gut Microflora and Secretion of Mucus *

doi: 10.1074/jbc.M114.632257

Figure Lengend Snippet: Secretion of colonic mucins in WT and Atg7 cKO mice. Hematoxylin and eosin (A), Alcian blue-PAS (B), and anti-MUC2 (C) staining of the mouse distal colon. D, percentage of mucus-secreting crypts. E, quantification of inner mucus layer thickness. F, detection of fecal MUC2 by ELISA. G, quantitative RT-PCR. The relative quantity of Muc2 mRNA compared with the quantity of β-actin was determined by the ΔΔCt method. Error bars represent the S.D. of samples within a group. Asterisk in A–C, mucus-secreting crypt. Arrows in A–C, mucus-secreting goblet cells. Dashed line in C, mucosal surface. An unpaired Student's t test was used to compare WT versus cKO mice. **, p < 0.01; *, p < 0.05. n = 3 per group.

Article Snippet: After blocking with PBS containing 3% BSA, 0.5 μg/ml rabbit anti-mouse MUC2 polyclonal antibody (H-300; Santa Cruz Biotechnology) or IgG from rabbit serum (Sigma) was added to the wells and incubated overnight.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Medicine

Article Title: Intestinal changes in permeability, tight junction and mucin synthesis in a mouse model of Alzheimer's disease

doi: 10.3892/ijmm.2023.5316

Figure Lengend Snippet: Monoclonal primary antibodies in western blotting.

Article Snippet: Sections were immunostained with a rabbit anti-MUC2 antibody (1:100; red; cat. no. #DF8390; Affinity Biosciences) and incubated at 4°C overnight.

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Intestinal changes in permeability, tight junction and mucin synthesis in a mouse model of Alzheimer's disease

doi: 10.3892/ijmm.2023.5316

Figure Lengend Snippet: Variation in number of GCs and MUC2 expression in intestinal mucosa. (Aa-h) Alcian blue-Periodic acid Schiff staining showed GCs and (Ba-h) immunohistochemistry showed MUC2 in the ileum and proximal colon of WT and dTg mice at 6 and 12 months. (C) Number of GCs, as determined by histochemical staining of the ileum and proximal colon. (D) MUC2 expression area in the ileum and proximal colon was determined by immunohistochemistry (n=6/group). Scale bar, 50 µ m. (E) Protein levels of MUC2 in extracts of intestinal tissue were detected by western blotting. (F) Quantification of the western blot results (n=4/group). * P<0.05, ** P<0.01. GC, goblet cell; WT, wild-type; dTg, double transgene; MUC2, mucin 2; WGA, wheat germ agglutin.

Article Snippet: Sections were immunostained with a rabbit anti-MUC2 antibody (1:100; red; cat. no. #DF8390; Affinity Biosciences) and incubated at 4°C overnight.

Techniques: Expressing, Staining, Immunohistochemistry, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Intestinal changes in permeability, tight junction and mucin synthesis in a mouse model of Alzheimer's disease

doi: 10.3892/ijmm.2023.5316

Figure Lengend Snippet: Variation in MUC2 and WGA in intestinal mucosa. (Aa-p) IF showed MUC2 and WGA in ileum of WT and dTg mice at 6 and 12 months. (B) MUC2- and (C) WGA-positive area was determined by IF in ileum (n=6/group). IF showed MUC2 and WGA in (Da-p) proximal colon of WT and dTg mice at 6 and 12 months. (E) MUC2- and (F) WGA-positive area was determined by IF in proximal colon (n=6/group). * P<0.05, ** P<0.01. WGA, wheat germ agglutinin; IF, immunofluorescence; MUC2, mucin 2; WT, wild-type; dTg, double transgene.

Article Snippet: Sections were immunostained with a rabbit anti-MUC2 antibody (1:100; red; cat. no. #DF8390; Affinity Biosciences) and incubated at 4°C overnight.

Techniques: Immunofluorescence

Journal: Gut Microbes

Article Title: Dietary modulation of gut microbiota affects susceptibility to drug-induced liver injury

doi: 10.1080/19490976.2024.2439534

Figure Lengend Snippet: WSD-induced gut microbiota dysbiosis impaired intestinal barrier function, facilitating LPS translocation to the liver and triggering hepatic inflammation. (a) Heatmap representing the log 2 -transformed absolute abundance of the ASVs that were significantly different between saline-treated mice in NC and WSD groups. (b) The absolute abundance of secondary bile acid biosynthesis-related genes predicted by PICRUSt2. (c) The concentrations of bile acids in the cecum content of mice. (d) Representative images of MUC2 and ZO-1 immunohistochemical staining in the colon (scale bar = 50 μm) and calculated average optical density (AOD). (e) Representative images of liver immunohistochemistry sections stained with LPS antibody (scale bar = 50 μm) and calculated LPS positive area (%). (f) GSEA-based KEGG enrichment plots of the top 10 significantly activated pathways in WSD, compared to NC. NES, normalized enrichment score. (g)Heatmap of scaled gene counts related to neutrophil extracellular trap formation and GSH synthesis based on RNA-Seq data. (h) The level of hepatic GSH of saline-treated mice in NC and WSD. For (b), (c), and (e), n = 7. For (d), n = 5. For (h), n = 6–7. Data are presented as the mean ± SEM and analyzed using two-tailed unpaired Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For immunohistochemistry staining of MUC2 and ZO-1, after deparaffinization and processing for antigen retrieval, the colon sections were incubated overnight at 4°C with anti-rabbit MUC2 antibody (1: 500, Cat# GB11344; Servicebio, China) anti-rabbit ZO-1 antibody (1: 200, Cat# GB111981; Servicebio, China).

Techniques: Translocation Assay, Transformation Assay, Saline, Immunohistochemical staining, Staining, Immunohistochemistry, RNA Sequencing, Two Tailed Test

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: Journal of Virology

Article Title: Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site

doi: 10.1128/JVI.01258-20

Figure Lengend Snippet: Nasal mucosal tissue transcriptional response to HCMV infection. RNA was extracted from mock- and HCMV-infected tissues at 24 hpi and subjected to transcriptome analysis. To account for the potential donors’ tissue-to-tissue variability, two pools (each representing a mixture of 5 independent donor tissues) for each experimental condition were used, together representing 10 tissues from different individuals. (A) MA plot (log ratio versus abundance) comparing gene expression profiles in mock- and HCMV-infected tissues for all 26,340 queried genes. Genes of higher abundance in infected tissues are indicated by positive changes, and those of lower abundance in infected tissues are indicated by negative changes. Red dots denote significant genes (adjusted P [Padj] < 0.1); orange dots denote added nonsignificant genes (see Materials and Methods). (B) Heat map representation of all differentially expressed and added genes (red and orange dots in panel A), comparing the two pools of mock- and HCMV-infected tissues. Normalized expression values were scaled at gene level (scale is shown at top-right), then hierarchically clustered and drawn as a heat map. Representative upregulated innate immunity genes further analyzed by qRT-PCR as shown in panel F and downregulated epithelial-cell related genes are indicated. (C) Selected antiviral and proinflammatory innate immunity genes with a strong (>3-fold) upregulation in HCMV-infected versus mock-infected tissues. (D) AQP5 and MUC2 mRNA expression levels in HCMV infected tissues relative to the normalized levels (normalized to a value of 1) in mock-infected tissues. (E and F) The indicated viral/cellular mRNA levels were analyzed by qRT-PCR and normalized by cellular β-actin. The fold change between HCMV-infected and mock-infected tissues (F) is shown for each cellular gene in 6 independent nasal turbinate tissues (T1 to T6) obtained from different individuals. (G and H) Functional analysis of conditioned medium (CM) recovered at 1 (E) and 5 (F) dpi from mock- and HCMV-infected nasal turbinate tissues (prepared as described in Materials and Methods). (G) Human foreskin fibroblasts (HFF) and human retinal pigmented epithelial cells (ARPE) cultures were pretreated overnight with conditioned medium (CM) and infected with HCMV. HCMV IE1 mRNA levels were analyzed by qRT-PCR at 24 hpi and normalized by cellular β-actin. (H) Peripheral blood leukocytes (PBL) transwell migration toward CM from mock- or HCMV-infected nasal turbinate cultures (treated with ganciclovir when indicated). CM samples were preincubated with no IgG (control) or with α-CXCL10 antibodies, as indicated. The number of migrated cells was determined by flow cytometry. The data shown are representative of at least three independent experiments. Significant changes are indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001. NT, nontreated; n.s., nonsignificant.

Article Snippet: Tissues were fixed in 2% paraformaldehyde for 30 min, washed in phosphate-buffered saline (PBS) and transferred to 80% ethanol, permeabilized by 1% Triton X-100 and treated with CAS-Block in order to avoid nonspecific antibody binding, followed by incubation with CAS-Block containing the following antibodies (or with CAS-Block alone as control) for specific cell markers: rabbit monoclonal antibodies (MAbs) against E-cadherin (dilution, 1:200; clone 24E10; Cell Signaling Technology, Inc.), mouse MAbs against MUC2 (dilution, 1:200; catalog no. ab11197; Abcam, Cambridge, UK), mouse MAbs against vimentin (dilution, 1:100; Dako), rabbit polyclonal antibodies (Abs) against von Willebrand factor (dilution, 1:800; Dako), and mouse MAbs against CD68 (dilution, 1:50; Abcam, Cambridge, United Kingdom) for the detection of: epithelial cells, goblet cells, stromal cells, endothelial cells, and macrophages, respectively.

Techniques: Infection, Expressing, Quantitative RT-PCR, Functional Assay, Migration, Flow Cytometry

Journal: Journal of Virology

Article Title: Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site

doi: 10.1128/JVI.01258-20

Figure Lengend Snippet: Primers and probes for real-time PCR analysis

Article Snippet: Tissues were fixed in 2% paraformaldehyde for 30 min, washed in phosphate-buffered saline (PBS) and transferred to 80% ethanol, permeabilized by 1% Triton X-100 and treated with CAS-Block in order to avoid nonspecific antibody binding, followed by incubation with CAS-Block containing the following antibodies (or with CAS-Block alone as control) for specific cell markers: rabbit monoclonal antibodies (MAbs) against E-cadherin (dilution, 1:200; clone 24E10; Cell Signaling Technology, Inc.), mouse MAbs against MUC2 (dilution, 1:200; catalog no. ab11197; Abcam, Cambridge, UK), mouse MAbs against vimentin (dilution, 1:100; Dako), rabbit polyclonal antibodies (Abs) against von Willebrand factor (dilution, 1:800; Dako), and mouse MAbs against CD68 (dilution, 1:50; Abcam, Cambridge, United Kingdom) for the detection of: epithelial cells, goblet cells, stromal cells, endothelial cells, and macrophages, respectively.

Techniques: Real-time Polymerase Chain Reaction, Sequencing, SYBR Green Assay

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