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: Porcine Health Management

Article Title: Dietary supplementation of Galactooligosaccharides improves growth performance and intestinal barrier function of offspring

doi: 10.1186/s40813-025-00437-6

Figure Lengend Snippet: Effect of maternal GOS supplementation on inflammatory profiles, sIgA level and MUC2 expression in piglets. A Relative expression level of cytokine mRNA in the ileum mucosa, B Relative expression level of cytokine mRNA in the colon mucosa, C The content of cytokines in serum. D The content of sIgA in the ileum mucosa, E Immunofluorescence of sIgA in the ileum, F Immunofluorescence of NLRP3 in the ileum G Relative expression levels of MUC2 mRNA in the Ileum mucosa, H Relative expression level of MUC2 mRNA in the colon mucosa, I Immunofluorescence of MUC2 in the ileum (scale bars: 50 μm). Values are presented as means ± SEM, n = 8, One-Way ANOVA, Different lowercase letters indicate significant differences amongst treatment treatments ( P < 0.05). Con, Basal diet; GOS, Basic diet with 0.5% Galactooligosaccharides (Purity of approximately 70%); AB, Basic diet with 100 ppm chloramphenicol, 20 ppm ciprofloxacin, and 10 ppm vancomycin

Article Snippet: The sections were then blocked with 5% BSA blocking solution at room temperature for 30 min. Primary antibodies targeting tight junction proteins (ZO-1, occludin) and mucin 2 (MUC2) (Aifang Biotechnology, Changsha, China), Immunoglobulin A (sIgA) (Proteintech, Wuhan Sanying, China), cell proliferation markers (Ki67), and inflammasomes (NLRP3) were applied and incubated overnight at 4°C.

Techniques: Expressing, Immunofluorescence

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: bioRxiv

Article Title: Phosphatase-independent suppression of mucosal inflammation and disease progression by Salmonella SopB

doi: 10.1101/2024.09.02.610793

Figure Lengend Snippet: (A) Small intestinal tissue sections stained with H&E of non-infected (n=3) neonates or neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. Bar=100 µm. (B) Quantification of the depth of the lamina propria as a measure of the tissue edema measured in 15-20 different areas of one tissue section per animal (non-infected neonates (n=3) or neonates infected with wt (n=3) or Δ sopB (n=3)). (C) Muc2 immunostaining (red) in small intestine tissue sections of non-infected (n=3) neonates and the neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. Counterstaining with WGA (white), DAPI (blue); autofluorescence (green). Bar=50 µm. (D) Percentage of goblet cells among intestinal epithelial cells in non-infected (n=3) neonates and neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. 10-20 images with the size of 312µm x 250µm were evaluated on one tissue section per animal. (E) Goblet cell size in tissue sections of uninfected (n=3) neonates and neonates infected with wt (n=3) or Δ sopB (n=3) S . Typhimurium at day 2 p.i.. 11-21 goblet cells (µm 2 ) were analyzed on each section. (F) TUNEL staining (red) of neonatal small intestine tissue sections of non-infected (n=2) neonates and neonates infected with wt (n=5) or Δ sopB (n=4) S . Typhimurium at day 2 p.i.. White boxes indicate enlarged images (i, ii). Counterstaining with DAPI (blue). Bar=50 µm; insert, 20 µm. (G) Number of TUNEL positive cells in tissue sections of uninfected (n=2) neonates and the neonates infected with wt (n=5) or Δ sopB (n=4) S . Typhimurium at day 2 p.i. 9 -12 images with the size of 624µm x 501µm were evaluated per animal. (H) Ki67 (red) immunostaining on small intestine tissue sections of the neonates infected with wt (n=3) or Δ sopB (n=4) S . Typhimurium at day 2 p.i.. Counterstaining with E-cadherin (green), WGA (white), and DAPI (blue). Bar=50 µm, insert: 20 µm. (I) Number of Ki67 positive cells in small intestinal tissue sections of uninfected (n=2) neonates and the neonates infected with wt (n=5) or Δ sopB (n=4) S . Typhimurium at day 2 p.i. 5-29 crypts were analyzed per section. Representative images are shown. Quantified data are shown as individual points in violin plots, solid lines represent the median. Data were analyzed using One-way ANOVA Kruskal–Wallis test with Dunn’s multiple comparisons post-test. ns, non-significant; *, p<0.05; ****, p<0.0001.

Article Snippet: Rabbit anti-Ki67 (Ab15580, Abcam), mouse anti-E-cadherin (610182, BD Transduction Laboratories), rat anti-PMN (Ly6-6B2, SeroTec), rabbit anti-Muc2 (GTX100664, BIOZOL) were used at the appropriate dilution followed by the fluorophore-conjugated donkey secondary antibody (Jackson ImmunoResearch).

Techniques: Staining, Infection, Immunostaining, TUNEL Assay

Journal: Evidence-based complementary and alternative medicine : eCAM

Article Title: Liangxue Tongyu Prescription Alleviates Brain Damage in Acute Intracerebral Hemorrhage Rats by Regulating Intestinal Mucosal Barrier Function.

doi: 10.1155/2022/2197763

Figure Lengend Snippet: Figure 6: Efect of LTP on MUC2, SIgA, and GPR43 expression in the colon tissue of AICH rats: (a) MUC2 expression, (b) SIgA expression, (c) GPR43 expression, and (d) WB electrophoretograms of MUC2, SIgA, and GPR43 in the colon tissue samples. ap < 0.05 vs. the blank and sham groups. bp < 0.05 vs. the model group. cp < 0.05 vs. the LTP group (n 3).

Article Snippet: Te following primary antibodies were used for western blotting: rabbit anti-ZO-1 (Termo Fisher Scientifc, 40–2200, 1 :1000), OCLN (Abcam, ab167161, 1 :1000), CLDN-5 (ABclonal, A10207, 1 :1000), CLDN-1 (Termo Fisher Scientifc, 71–7800, 1 :1000), SIgA (Bioss Biotech, bs-0645R, 1 :1000), MUC2 (Boster Biotech, BM5029, 1 :1000), GPR43 (Bioss Biotech, bs-13536R, 1 :1000), and a βactin antibody (ABclonal, ac026, 1 :10000).

Techniques: Expressing