duolink® in situ mounting medium with dapi  (Millipore)

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate attenuates ethanol‐induced gastric mucosal hemorrhage and oedema in vivo. (A) Establishment of ethanol‐induced gastric ulcer in rats by intragastric administration of anhydrous ethanol. (B) Flowchart of the animal experimental procedures. (C) Macroscopic appearance of the gastric mucosa in four groups. (D) Quantification of gastric damage expressed as the ulcer index (UI). The UI was calculated as follows: UI = 10 × (ulcerated area/total mucosal area). (E) Representative HE‐stained images of stomach tissues. Scale bar = 50 µm. (F) Histological evaluation of gastric lesions using a microscopic HE score (0–14). The score sums the severity of four features: inflammatory cells (0–3), mucosal edema (0–4), hemorrhage (0–4), and epithelial loss (0–3). (G) Representative CD31 IF staining images in gastric mucosa. Scale bar = 20 µm. (H and I) RT‐qPCR analysis of Ang1 and Et1 mRNA expression in gastric mucosa. Target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. (J and K) The nitrate levels of the serum and gastric mucosa in the four groups. Quantitative data are expressed as the mean ± SD. * p < 0.05, *** p < 0.001, and ns denotes no significance. HE, hematoxylin–eosin; Nit, nitrate; IF, immunofluorescence; CD31, platelet endothelial cell adhesion molecule‐1; Ang1, angiotensin‐1; Et1, endothelin‐1; RT‐qPCR, real‐time quantitative polymerase chain reaction; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; SD, standard deviation.

Article Snippet: Coverslips were then mounted using the Duolink in situ mounting medium with DAPI (DUO82040; Merck) and sealed with clear nail polish.

Techniques: In Vivo, Staining, Quantitative RT-PCR, Expressing, Targeted Gene Expression, Control, Immunofluorescence, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate stabilizes the gastric mucus layer and improves mucosal healing of ethanol‐induced injury in vivo. (A) Representative gastric tissue images of AB–PAS staining. Scale bar = 50 µm. (B) Mucin histochemical analysis of gastric tissue. The mucin area was expressed as fold change relative to the control group. (C) IF staining of Tgf‐β1 (green) and DAPI (blue). (D) IF analysis of Tgf‐β1 with MFI. Scale bar = 200 µm. (E) RT‐qPCR analysis of Tgfb1 mRNA expression in gastric mucosa. Target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. (F) IF staining of Vegf‐a (red) and DAPI (blue). Scale bar = 200 µm. (G) IF analysis of Vegf‐a with MFI. (H) RT‐qPCR analysis of Vegfa mRNA expression in gastric mucosa. Target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. Quantitative data are expressed as the mean ± SD. * p < 0.05, *** p < 0.001, and ns denotes no significance. IF, immunofluorescence; Nit, nitrate; RT‐qPCR, real‐time quantitative polymerase chain reaction; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; AB–PAS, alcian blue and periodic acid‐Schiff; Vegf‐a, vascular endothelial growth factor a; Tgf‐β1, transforming growth factor beta 1; MFI, mean fluorescence intensity; SD, standard deviation.

Article Snippet: Coverslips were then mounted using the Duolink in situ mounting medium with DAPI (DUO82040; Merck) and sealed with clear nail polish.

Techniques: In Vivo, Staining, Control, Quantitative RT-PCR, Expressing, Targeted Gene Expression, Immunofluorescence, Real-time Polymerase Chain Reaction, Fluorescence, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate reduces ethanol‐induced inflammatory response in vivo. (A) IF staining of Tnf‐α (green) and DAPI (blue). (B) IF analysis of Tnf‐α with MFI. Scale bar = 200 µm. (C) RT‐qPCR analysis of Tnfa mRNA expression of the gastric mucosa. Target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. (D) IF staining of Il‐1β (red) and DAPI (blue). Scale bar = 200 µm. (E) IF analysis of Il‐1β with MFI. (F) RT‐qPCR analysis of Il1b mRNA expression in gastric mucosa. The target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01. IF, immunofluorescence; Nit, nitrate; RT‐qPCR, real‐time quantitative polymerase chain reaction; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Tnf‐a, tumor necrosis factor alpha; Il‐1β, interleukin‐1β; MFI, mean fluorescence intensity; SD, standard deviation.

Article Snippet: Coverslips were then mounted using the Duolink in situ mounting medium with DAPI (DUO82040; Merck) and sealed with clear nail polish.

Techniques: In Vivo, Staining, Quantitative RT-PCR, Expressing, Targeted Gene Expression, Control, Immunofluorescence, Real-time Polymerase Chain Reaction, Fluorescence, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate upregulates Tff2. (A) Venn diagram showing the unique and overlapping genes presented in paired groups and GU‐related potential targets. (B) Representative immunoblotting band of Tff2 and Mlc protein. (C and D) Analyses of immunoblotting band gray value in (B). (E and F) Representative Tff2 IF staining images in gastric mucosa and MFI score. Scale bar = 100 µm. (G) RT‐qPCR analysis of Tff2 mRNA expression in gastric mucosa. Target gene expression was normalized to Gapdh mRNA and expressed as fold change relative to the control group. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. GU, gastric ulcer; Nit, nitrate; Mlc, myosin light chain 2; pMlc, phosphorylated myosin light chain 2; IF, immunofluorescence; RT‐qPCR, real‐time quantitative polymerase chain reaction; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; MFI, mean fluorescence intensity; Tff2; trefoil factor 2; SD, standard deviation.

Article Snippet: Coverslips were then mounted using the Duolink in situ mounting medium with DAPI (DUO82040; Merck) and sealed with clear nail polish.

Techniques: Western Blot, Staining, Quantitative RT-PCR, Expressing, Targeted Gene Expression, Control, Immunofluorescence, Real-time Polymerase Chain Reaction, Fluorescence, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate promotes migration by TFF2 upregulation in vitro. (A) Images of the scratch healing process of GES‐1 cells in Ibidi culture inserts. Scale bar = 500 µm. (B and C) Quantitative analysis of the migration rate at 24 h and 48 h in (A). (D and E) Representative immunoblotting band of the TFF2 protein and analysis of band gray values. (F) IF staining of pMLC (pink) and DAPI (blue). Scale bar = 40 µm. (G) IF analysis of pMLC with MFI. (H) Images of the scratch healing process of GES‐1 cells transfected with si‐ TFF2 /si‐negative control in Ibidi culture‐inserts. Scale bar = 500 µm. (I and J) Quantitative analysis of the migration rate at 24 h and 48 h. (K) Images of the scratch healing process of GES‐1 cells transfected with si‐ TFF2 /si‐negative control in Ibidi culture‐inserts containing ethanol. Scale bar = 500 µm. (L and M) Quantitative analysis of the migration rate at 24 h and 48 h. Migration rate is quantified by the percentage of closed area to the initial scratch area. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and ns denotes no significance. TFF2, trefoil factor 2; GES‐1, human gastric epithelial; EtOH, ethanol; Nit, nitrate; Ctrl, control; MLC, myosin light chain 2; pMLC, phosphorylated myosin light chain 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; NC, negative control; SD, standard deviation.

Article Snippet: Coverslips were then mounted using the Duolink in situ mounting medium with DAPI (DUO82040; Merck) and sealed with clear nail polish.

Techniques: Migration, In Vitro, Western Blot, Staining, Transfection, Negative Control, Control, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

Article Snippet: Coverslips were then mounted using the Duolink in situ mounting medium with DAPI (DUO82040; Merck) and sealed with clear nail polish.

Techniques: Inhibition, In Vitro, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Autoradiography, Labeling, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Negative Control, Staining, Western Blot, Targeted Gene Expression, Plasmid Preparation, Standard Deviation, Proximity Ligation Assay