dapi mounting media  (SouthernBiotech)

Journal: bioRxiv

Article Title: Modified meiosis in the tardigrade Hypsibius exemplaris maintains heterozygosity across the genome

doi: 10.64898/2026.03.11.711151

Figure Lengend Snippet: (a) Each dot represents a tardigrade embryo that was fixed along a timeline from 0 to 125 minutes post-laying (mpl). Dots to the left of zero on the timeline represent those that were cut out of gravid adults directly into fixative solution. Embryos were then stained with DAPI and imaged using a scanning confocal microscope. During analysis, embryos were categorized into one of five groups depending on the number and orientation of visible chromosomes. Uncategorized embryos are shown in gray. Representative images of DAPI staining within embryos, one for each category, are shown. The arrow points to a polar body. Scale bars are 5um. (b) Live embryos were imaged every 5min with Differential Interference Contrast (DIC), and the relative timing of when they were laid was recorded. Each line indicates the timeline of a single embryo progressing through meiosis, starting from the time it was laid. Orange squares indicate the first time DNA migrated to the periphery, and green diamonds indicate the second time DNA migrated to the periphery. Blue circles indicate the first time a polar body was visible (characterized by a membrane surrounding DNA at the periphery). Only embryos where a polar body was ultimately visible were included. Finally, images of a single representative embryo are shown over time as it proceeds through meiosis, with symbols marking the events corresponding to the above timelines. The arrow points to a polar body. Scale bars are 5um. (c) Model of modified meiosis cytology in H. exemplaris . Inferred homologous chromosomes are differentially colored, purple and orange. Light gray surrounding the chromosomes represents the area visible by DIC imaging. Meiosis I metaphase and anaphase occur but do not conclude with polar body cytokinesis. Meiosis II then proceeds as normal, resulting in a single polar body. Alt text: Timelines ranging from 0 to 130 minutes post-laying, symbols depicting replicates of imaged H. exemplaris embryos, and representative images for different phases of modified meiosis are shown for each of two microscopy experiments (panels a and b).

Article Snippet: Embryos were then mounted onto slides with 28.41μm glass beads (Whitehouse Scientific MS0028) and approximately 2uL of DAPI fluoromount-G mounting media (Southern Biotech 0100-20).

Techniques: Staining, Microscopy, Membrane, Modification, Imaging

Journal: bioRxiv

Article Title: Epithelial sensing of vitamin A shapes intestinal antimicrobial defense

doi: 10.64898/2026.03.08.710399

Figure Lengend Snippet: (A) Vitamin A deprivation model. Pregnant dams were fed vitamin A–deficient (Vit A–) or vitamin A–replete (Vit A+) diets during gestation. Offspring were maintained on the maternal diet for at least 8 weeks. (B) Single cell RNA sequencing (scRNA-seq) was performed on a single cell suspension from the mouse small intestine. Live cells were bar-coded and sequenced, and cells annotated as epithelial cells were analyzed and are displayed as a two-dimensional reduction plot (UMAP). Both Vit A+ and Vit A– cells are plotted (n=3 mice per group). (C) Distribution of epithelial cell subsets from Vit A+ and Vit A– mice (n=3) projected on the same UMAP as in (B). (D) Heatmap of average normalized expression of known vitamin A-responsive genes from the scRNA-seq analysis of IECs. (E) Volcano plot of vitamin A-dependent changes in gene expression in IECs analyzed by scRNA-seq. Reg3b and Reg3g are highlighted in red. (F) Density plots of Reg3b and Reg3g expression in IECs analyzed by scRNA-seq. (G) The gut microbiota induces expression of the antibacterial proteins REG3β and REG3γ. Microbial molecular patterns activate a dendritic cell–ILC3 signaling relay that drives IL-22 production, which induces Reg3b and Reg3g expression in IECs. (H) qPCR analysis of Reg3g and Reg3b transcript abundance in small intestines from conventional mice fed Vit A+ (n=12) and Vit A– (n=14) diets, and germ-free mice fed a Vit A+ diet (n=9). Mice were from three independent litters. Each data point represents one mouse. (I) Immunofluorescence microscopy of REG3G in the distal small intestine of Vit A+ or Vit A– mice. Sections were stained for REG3G (red) and counterstained with DAPI (blue). Scale bar, 100 μm. Images are representative of three mice per group. (J) Mean fluorescence intensities of at least 65 villi were determined across three mice in each dietary group. Vit A+, vitamin A+; Vit A–, vitamin A–; wk, weeks; scRNA-seq, single cell RNA sequencing; IEC, intestinal epithelial cell; UMAP, Uniform Manifold Approximation and Projection; qPCR, quantitative real-time PCR; REG3B, regenerating islet-derived protein 3β; REG3G, regenerating islet-derived protein 3γ; Conv, conventional; GF, germ-free. Means ± SEM are plotted; ***p < 0.001 by Mann-Whitney test. See also .

Article Snippet: Slides were washed three times with PBS and mounted using Fluoromount-G Mounting Media with DAPI (SouthernBiotech 0100-20) to label nuclei.

Techniques: Single Cell, RNA Sequencing, Suspension, Expressing, Gene Expression, Immunofluorescence, Microscopy, Staining, Fluorescence, Real-time Polymerase Chain Reaction, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epithelial sensing of vitamin A shapes intestinal antimicrobial defense

doi: 10.64898/2026.03.08.710399

Figure Lengend Snippet: (A) Mass spectrometry measurement of retinol from serum of mice fed provided a Vit A+ (n=9) or Vit A– (n=14) diet as described in . Each dot represents one mouse, and the limit of detection (L.O.D.) is indicated. (B) Expression of marker genes among small intestinal cell populations as determined by scRNA-seq. (C) Immunofluorescence microscopy of REG3G in small intestine sections from Vit A+ mice, with comparison to an isotype control antibody. Sections were stained for REG3G (red) and counterstained with DAPI (blue) to detect nuclei. Scale bar, 100 μ m. (D) Representative images of small intestinal crypts of mice fed a Vit A+ or Vit A– diet. Paneth cells were identified by their distinctive morphology and the presence of dense secretory granules (white arrowheads). Scale bar, 50 μ m. (E) Enumeration of Paneth cells identified in crypts from Vit A+ (60 crypts counted across 5 mice) and Vit A–mice (94 crypts counted across four mice). (F) Immunofluorescence microscopy of lipid A (bacteria) and UEA-I (mucus) in small intestine sections from Vit A+ mice and Vit A- mice. The tissue was counterstained with DAPI to detect nuclei. The mucus barrier is outlined with a white dotted line and examples of positive lipid A staining are indicated by white arrow heads. Vit A, vitamin A; L.O.D., limit of detection; REG3G, regenerating islet-derived protein 3γ; Ctrl, control; UEA-I, Ulex Europaeus Agglutinin I. Means ± SEM are plotted; ns, not significant by Mann-Whitney test.

Article Snippet: Slides were washed three times with PBS and mounted using Fluoromount-G Mounting Media with DAPI (SouthernBiotech 0100-20) to label nuclei.

Techniques: Mass Spectrometry, Expressing, Marker, Immunofluorescence, Microscopy, Comparison, Control, Staining, Bacteria, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epithelial sensing of vitamin A shapes intestinal antimicrobial defense

doi: 10.64898/2026.03.08.710399

Figure Lengend Snippet: (A) HT-29 cells were treated with 1 μ M retinol and/or 100 ng/mL IL-22. REG3G transcripts were quantified by qPCR 18 hours later. Each data point represents one experimental replicate (n=6 per group). (B) Retinol is converted to RA through a two-step enzymatic reaction catalyzed by retinol/alcohol dehydrogenases and retinaldehyde/aldehyde dehydrogenases. Disulfiram inhibits aldehyde dehydrogenase enzymatic activity (including RALDH). (C) HT-29 cells were treated with 1 μ M RA and/or 100 ng/mL IL-22 in the presence of vehicle or 100 μ M disulfiram. REG3G transcripts were quantified by qPCR after 18 hours. Each data point represents an independent experimental replicate (n=6 per group). (D) qPCR analysis of REG3G transcripts in HT-29 cells treated with retinol and IL-22 in the presence or absence of disulfiram. Each data point represents one experimental replicate (n=4 per group). (E) qPCR analysis of REG3G transcripts in HT-29 cells treated with retinol and IL-22 in the presence of disulfiram, with rescue by RA. Each data point represents an independent experimental replicate (n=4 per group). (F) The Rdh7 -/- mouse carries a global deletion of the gene encoding RDH7, which catalyzes the first step in the retinol-to-RA conversion. Rdh7 μIEC mice harbor an IEC–specific deletion of Rdh7 , generated by crossing Rdh7 fl/fl mice with Villin-Cre transgenic mice. (G) qPCR analysis of Reg3g transcripts in the small intestines of conventional wild-type (n=5) and Rdh7 -/- (n=5) mice, and germ-free wild-type (n=21) mice. Each data point represents one mouse. (H) Immunoblot of REG3G in small intestines from conventional wild-type (n=3) and Rdh7 -/- (n=3) mice. ACTIN was the loading control. Each lane is from one mouse. (I) qPCR analysis of Reg3g transcripts in small intestines from six litters of conventional Rdh7 fl/fl (n=11) and Rdh7 μIEC (n=13) mice and germ-free wild-type (n=21) mice. Each data point represents one mouse. (J) Rdh7 μIEC mice received two intraperitoneal injections of vehicle or retinoic acid (1 μ M), administered 12 hours apart. Mice were sacrificed 12 hours after the last injection. (K) qPCR analysis of Reg3g transcripts in the intestines of two litters of Rdh7 fl/fl (n=3) and Rdh7 μIEC (n=6) littermates injected intraperitoneally with RA or vehicle. Each data point represents one mouse. (L) Immunofluorescence microscopy of REG3G in the small intestines of Rdh7 fl/fl and Rdh7 μIEC littermates injected via the intraperitoneal route with retinoic acid (RA) or vehicle. Sections were stained for REG3G (red) and counterstained with DAPI (blue). Scale bar, 100 μm. Images are representative of at least three fields per sample from two independent experiments (three littermates per group). (M) Mean fluorescence intensities of at least 150 villi from the images represented in (L) were quantified across at least two mice in each experimental group. IEC, intestinal epithelial cell; REG3G, regenerating islet-derived protein 3γ; qPCR, quantitative real-time PCR; RDH, retinol dehydrogenase; RALDH, retinaldehyde dehydrogenase; Conv, conventional; GF, germ-free; i.p., intraperitoneal; RA, retinoic acid. Means ± SEM are plotted; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant by Mann-Whitney test. See also .

Article Snippet: Slides were washed three times with PBS and mounted using Fluoromount-G Mounting Media with DAPI (SouthernBiotech 0100-20) to label nuclei.

Techniques: Activity Assay, Generated, Transgenic Assay, Western Blot, Control, Injection, Immunofluorescence, Microscopy, Staining, Fluorescence, Derivative Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epithelial sensing of vitamin A shapes intestinal antimicrobial defense

doi: 10.64898/2026.03.08.710399

Figure Lengend Snippet: (A) Vitamin A-derived retinol is metabolized to RA, which activates retinoic acid receptors (RARs). RARs bind target gene promoters to drive RA-dependent transcription. (B) HT-29 cells (human intestinal epithelial cells) were treated with the RAR antagonist BMS493 or the RAR agonist Ch55 and simultaneously stimulated overnight with RA and IL-22. REG3G transcripts were quantified by qPCR. Each data point represents an independent experimental replicate (n=6 per group). (C) HCT-116, a transfection-competent human intestinal epithelial cell line expressing RARA and RARG , was treated for 24 hours with an siRNA targeting either gene and then stimulated overnight with retinol and IL-22. REG3G transcripts were quantified by qPCR. Each data point represents an independent experimental replicate (n=4 per group). (D) IEC-specific disruption of RAR signaling using a dominant-negative RAR (dnRAR) knock-in allele. dnRAR mice harbor a loxP -flanked STOP cassette upstream of a dominant-negative RAR open reading frame. The dnRAR is derived from a mutant human RARα (RAR403) lacking the ligand-dependent transactivation domain and functions as a pan-RAR inhibitor. Crossing dnRAR mice with Villin-Cre transgenic mice excises the STOP cassette in IECs, resulting in IEC-selective expression of dnRAR and inhibition of RAR signaling. (E) qPCR analysis of Reg3g expression in small intestines of conventional dnRAR fl/fl (n=12) and dnRAR IEC (n=17) mice from five litters, and germ-free wild-type mice (n=21). (F) Immunofluorescence microscopy of REG3G in small intestines of dnRAR fl/fl and dnRAR IEC mice. Sections were stained for REG3G and counterstained with DAPI. Scale bar, 100 μ m. Images are representative of at least three fields per sample and two independent experiments (three littermates per group). (G) Mean fluorescence intensities of at least 150 villi from the images represented in (F) were quantified across at least two mice of each genotype. RAR, retinoic acid receptor; RA, retinoic acid; IEC, intestinal epithelial cell; REG3G, regenerating islet-derived protein 3γ; siRNA, small interfering RNA; dnRAR, dominant negative retinoic acid receptor; Conv, conventional; GF, germ-free. Means ± SEM are plotted; *p < 0.05; **p < 0.01; ***p<0.001; ns, not significant by Mann-Whitney test. See also .

Article Snippet: Slides were washed three times with PBS and mounted using Fluoromount-G Mounting Media with DAPI (SouthernBiotech 0100-20) to label nuclei.

Techniques: Derivative Assay, Transfection, Expressing, Disruption, Dominant Negative Mutation, Knock-In, Mutagenesis, Transgenic Assay, Inhibition, Immunofluorescence, Microscopy, Staining, Fluorescence, Small Interfering RNA, MANN-WHITNEY