roti-mount fluor-care dapi  (Carl Roth GmbH)

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The impact of disease and species differences on the intestinal CLCA4 gene expression

doi: 10.1007/s00109-025-02538-9

Figure Lengend Snippet: A In adult mice, both Clca4a and - 4b are expressed on mRNA level in the small intestine, whereas only Clca4a is expressed in the large intestine. Relative expression levels of Clca4a and - 4b were determined by RT-qPCR with Ef- 1α , ß2M , and GAPDH as reference genes. Neither Clca4a nor - 4b were expressed in the stomach. In the small intestine, Clca4a and - 4b were expressed to a comparable degree. In the large intestine, Clca4b was not expressed whereas Clca4a was highly expressed, particularly in the caecum. Data are expressed as mean ± SEM and expression levels are provided in ratios of Clca to the reference genes Ef- 1α , ß2M , and GAPDH . Ct = cycle threshold, prox. = proximal, mid. = middle, dist. = distal. Adult mice; n = 4. B The expression of Clca4a and - 4b varies along the crypt-villus axis. The mRNA expression of Clca4a and - 4b was quantified by RT-qPCR in laser capture microdissected samples from specific intestinal locations. Both homologs were found expressed in the jejunal villi, whereas only Clca4b was also expressed in the jejunal crypts. In the colon, Clca4a was exclusively detected in the luminal areas but not in the crypts. Using this technique, traces of Clca4b transcripts were detected in both colon locations investigated. Data are expressed as mean ± SEM and expression levels are given in ratios of Clca to reference the genes Ef- 1α , ß2M , and GAPDH . Ct, cycle threshold. Adult mice; n = 3. C Clca4a and - 4b were expressed in intestinal epithelial cells. mRNA of both Clca4a (red, top panel) and - 4b (red, bottom panel) was detected in enterocytes of the jejunum via ISH. In contrast, only Clca4a but not Clca4b was detected in enterocytes of the large intestine. The stomach was devoid of Clca4a or - 4b expression. Adult mice; n = 3. Blue = Roti®-Mount Fluor-Care DAPI nucleus staining. Scale bars = 50 μm. D Clca4a and - 4b showed a regionally shifted expression pattern in enterocytes of the jejunal villi. Along the crypt-villus axis, Clca4a (red) and - 4b (blue) were both expressed by the same enterocytes, however, only in the central and the apical areas of the villi. In contrast, enterocytes of the villi below these areas exclusively expressed Clca4b (blue). Duplex ISH; adult mouse. Scale bar = 50 μm

Article Snippet: Amplifier and label probe hybridizations were performed using fast red as chromogen, followed by counterstaining with hematoxylin for 45 s, washing in tap water for 5 min, and mounting with Roti-Mount Fluor-Care DAPI (4, 6-diaminidino- 2-phenylindole; Carl Roth).

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The impact of disease and species differences on the intestinal CLCA4 gene expression

doi: 10.1007/s00109-025-02538-9

Figure Lengend Snippet: A Abundant de novo expression of Clca4b in colon tissue of a murine DSS colitis model. The expression of Clca4a and - 4b was quantified in proximal and distal colon of a murine DSS colitis model by RT-qPCR. Left – Clca4a expression did not show any difference between DSS colitis and healthy controls in the proximal colon. However, Clca4a was significantly upregulated in the distal colon under conditions of DSS colitis. Right – Clca4b expression significantly increased in proximal and also in distal colon in DSS colitis. Data are expressed as mean fold change and Ef- 1α , ß2M , and GAPDH were used as reference genes. Ct, cycle threshold. Dotted lines indicate fold changes of 0.5 and 2 as limits for valid statement of lowered or elevated expression, respectively. n = 10 per group. * p < 0.05 and **** p < 0.0001 vs. Ctrl and #### p < 0.0001 vs. proximal colon by Mann–Whitney-U test. B Clca4a and - 4b were markedly expressed in enterocytes of DSS-challenged mice. Clca4a (red, left panel) was expressed in the distal colon of DSS-challenged mice compared to healthy controls as determined via ISH. Clca4b (red, right panel) was also strongly expressed in both proximal and distal colon whilst being absent from healthy controls. The most prominent signals were detected in enterocytes adjacent to acute ulcerations (*). Murine DSS colitis model; n = 3. Blue = Roti®-Mount Fluor-Care DAPI nucleus staining. Scale bars = 20 μm

Article Snippet: Amplifier and label probe hybridizations were performed using fast red as chromogen, followed by counterstaining with hematoxylin for 45 s, washing in tap water for 5 min, and mounting with Roti-Mount Fluor-Care DAPI (4, 6-diaminidino- 2-phenylindole; Carl Roth).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Staining

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The impact of disease and species differences on the intestinal CLCA4 gene expression

doi: 10.1007/s00109-025-02538-9

Figure Lengend Snippet: A De novo expression of Clca4b in whole tissue lysates of a colitis-associated colon cancer (CAC) model. Clca4a expression levels (left panel) failed to show any statistically significant differences between neoplastic or non-neoplastic intestinal regions of the CAC model and healthy controls, although faintly downregulated in non-neoplastic CAC colon samples, as determined by RT-qPCR. Clca4b expression (right panel) was significantly increased in the neoplastic CAC colon. Data are expressed as mean fold change and Ef- 1α , ß2M , and GAPDH were used as reference genes. Ct, cycle threshold. n = 3 to 6 per group. ** p < 0.01 vs. Ctrl by unpaired two-tailed t-test. B In the colitis-associated colon cancer (CAC) model, Clca4a and - 4b were predominantely expressed in enterocytes at the junction between tumor and non-neoplastic area, but not in the tumor itself. In most animals, no expression of Clca4a or - 4b was detected via fluorescence ISH in the center of the neoplastic tissue. Clca4a (red, top panel) was moderately to strongly expressed at the tumor surface and border and only minimally to moderately in the tumor-adjacent non-neoplastic area. Clca4b (red, bottom panel) was not expressed at the tumor surface, however, it showed a moderate to strong expression at the border and a moderate expression in the non-neoplastic adjacent area. Murine CAC model, n = 5. Blue = Roti®-Mount Fluor-Care DAPI nucleus staining. Scale bars = 20 μm. Lower panels show the corresponding bright field images of the sections, red signals of chromogenic ISH = Clca4a and - 4b , respectively

Article Snippet: Amplifier and label probe hybridizations were performed using fast red as chromogen, followed by counterstaining with hematoxylin for 45 s, washing in tap water for 5 min, and mounting with Roti-Mount Fluor-Care DAPI (4, 6-diaminidino- 2-phenylindole; Carl Roth).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Fluorescence, Staining

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: The impact of disease and species differences on the intestinal CLCA4 gene expression

doi: 10.1007/s00109-025-02538-9

Figure Lengend Snippet: A Human CLCA4 mRNA was predominantly detected in colonic enterocytes. Previously published sc-RNA seq data were used to calculate the percentage of CLCA4 + cells per cell population. B Human CLCA4 mRNA was expressed in enterocytes of the large but not small intestine. ISH revealed a moderate, patchy CLCA4 expression (red) in luminal enterocytes of the large intestinal epithelium, whereas the small intestine lacked CLCA4 expression. Adult humans; n = 3. Blue = Roti®-Mount Fluor-Care DAPI nucleus staining. Scale bars = 20 μm. C In CD, an increase in frequency of CLCA4 + cells was found for all cell populations in the colon but only for enterocytes in the ileum. The same dataset as in A was used to calculate the percentage of CLCA4 + cells in healthy individuals and CD patients. D Cellular expression pattern of CLCA4 in colorectal cancer. Only scattered CLCA4 signals were found in the tumor cells by fluorescence ISH. In contrast, moderate to strong patchy signals were observed in the non-neoplastic epithelium adjacent to the tumor and, in particular, at the junction between tumor and healthy tissue. Adult humans; n = 3. Blue = Roti.®-Mount Fluor-Care DAPI nucleus staining. Scale bars = 20 μm. Lower panel shows the corresponding bright field images of the sections, red signals of chromogenic ISH = CLCA4 . E In human CRC, increased expression of CLCA4 was found in non-malignant enterocytes from tumorous tissue samples compared to enterocytes from adjacent healthy tissue. Tumor cells failed to show CLCA4 expression. Mean CLCA4 expression was calculated per cell cluster; data were taken from a previously published sc-RNA seq dataset of human CRC samples

Article Snippet: Amplifier and label probe hybridizations were performed using fast red as chromogen, followed by counterstaining with hematoxylin for 45 s, washing in tap water for 5 min, and mounting with Roti-Mount Fluor-Care DAPI (4, 6-diaminidino- 2-phenylindole; Carl Roth).

Techniques: RNA Sequencing, Expressing, Staining, Fluorescence

Journal: Virus Research

Article Title: Molecularly generated rat hepatitis E virus strains from human and rat show efficient replication in a human hepatoma cell line

doi: 10.1016/j.virusres.2024.199364

Figure Lengend Snippet: Characterization of HuH-7-Lunet BLR cells persistently infected with ratHEV strains R63 or pt2, or HEV genotype 3 (GT3) strain 47832mc. The cells were generated by splitting at 120 days after transfection with the respective genomic RNA. (A) Detection of viral RNA. The culture supernatant was analysed by RT-qPCR at the indicated time-points. ND: not detectable. (B) Detection of viral antigen. The cells were stained with the monoclonal antibody 9C8 directed against the HEV capsid protein and visualized by immunofluorescence (green). Cellular nuclei were stained with DAPI (blue). Scale bar: 20 µm. (C) Detection of viral particles. The culture supernatant was analyzed by transmission electron microscopy after negative staining with uranyl acetate. Scale bar: 100 nm.

Article Snippet: The cells were mounted with Roti®-Mount Fluor Care DAPI (Carl Roth, Karlsruhe, Germany) and analyzed using an Axio Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany).

Techniques: Infection, Generated, Transfection, Quantitative RT-PCR, Staining, Immunofluorescence, Transmission Assay, Electron Microscopy, Negative Staining

Journal: iScience

Article Title: Tonic activin signaling shapes cellular and synaptic properties of CA1 neurons mainly in dorsal hippocampus

doi: 10.1016/j.isci.2023.108001

Figure Lengend Snippet: Disruption of activin receptor signaling reduces basal synaptic transmission throughout hippocampus, but strongly augments dorsal short-term facilitation at 5 Hz (A) Schematic illustration of hippocampal slice, indicating sites of stimulation and recording in CA1 stratum radiatum. The illustration on the right side shows a representative field potential trace, consisting of stimulus artifact (truncated), fiber volley (FV), and postsynaptic potential (fPSP). (B) Input-output curves, where fPSP peak amplitude is plotted against stimulus intensity in dorsal CA1 (closed circles) and ventral CA1 (open circles) from wt (black) and mutant mice (red). (C and D) Hippocampal cultures (DIV 20–25) were stained for VGLUT1 (green), MAP2 (red) and DAPI (blue) to visualize synaptic vesicle transporter expression on identified neuronal neurites (C) with or without recombinant activin A (25 ng/mL, 24 h). Scale bar, 20 μm. Histogram of VGLUT1/MAP2 ratio (D) shows no significant effect of activin A on VGLUT1 expression. (E) Paired-pulse stimuli were delivered at inter-stimulus intervals (ISI) ranging from 10 ms up to 500 ms in dorsal CA1 (closed circles) and ventral CA1 (open circles). PPRs in both areas were not affected by lack of activin signaling. Inset in E from a wt slice shows representative PPR at 50 ms ISI. (F) Hippocampal slices from wt (black) and dnActRIB (red) mice containing dorsal CA1 (closed circles) or ventral CA1 (open circles) were stimulated with 100 pulses at 5 Hz. Last 10 pulses were averaged and used for statistical comparison. Values are shown as mean ± SEM. Statistical comparisons were performed using an unpaired, two-tailed Student’s t test at α = 0.05 (B, E, and F) or a one-way ANOVA followed by Tukey’s post-hoc test (D). ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: After washing with PBS (thrice, 10 min), coverslips were mounted with Roti®Mount Fluor Care DAPI (Carl Roth, Karlsruhe, Germany).

Techniques: Disruption, Transmission Assay, Mutagenesis, Staining, Expressing, Recombinant, Comparison, Two Tailed Test