epifluorescence microscope nikon eclipse ti-2  (Nikon)

Journal: Nature Communications

Article Title: Efficient generation of germline chimeras in a non-rodent species using rabbit induced pluripotent stem cells

doi: 10.1038/s41467-025-60314-2

Figure Lengend Snippet: a Experimental scheme of B19_VAL, B19_VAL_14d, NaiveRep_KF, and NaiveRep_VAL cell line generation. b Immunostaining for SOX2, OCT4, DPPA5, OOEP, 5mC, H3K9me3, H3K14ac, and H3K4me3 in B19 cells before (_KF) and after (_VAL) switching to VALGöX culture medium. Scale bars: 50 µm for immunostainings, 100 µm for phase contrast. Violin plots show fluorescence intensity distribution. Means and standard deviations were calculated from measurements in at least 1,000 cells per condition (exact n indicated for each, from independent experiments: 3 for OCT4, OOEP, DPPA5, and 5mC; 4 for SOX2; 5 for H3K14ac and H3K4me3; 6 for H3K9me3). Comparisons between KF and VAL conditions were made using a two-sided Welch’s t -test for unequal variances. No significant differences were observed for SOX2 and H3K4me3 (difference between means <10%) and OCT4 ( p = 0.35). Source data are provided in the Source Data file. c Heatmap of differentially expressed genes between B19_KF and B19_VAL cells (three biological replicates). d Phase-contrast images of B19 cells 48 h (_VAL) and 14 days (_VAL_14d) after transfer to VALGöX culture medium (5 experiments). Scale bars: 200 µm. e Heatmap of differentially expressed genes between B19_VAL and B19_VAL_14d cells. f Phase-contrast and epifluorescence images of NaiveRep_KF and NaiveRep_VAL cells (5 experiments). Scale bars: 100 µm. g Flow cytometry analysis of parental B19_KF, NaiveRep_KF, and NaiveRep_VAL showing blue and red fluorescence corresponding to EOS-tagBFP and DDPA2-mKO2, respectively. Basal tagBFP expression in NaiveRep_KF cells reflects endogenous ETn promoter activity downstream of the naïve-state-specific distal enhancer of OCT4/POU5F1 in the EOS vector .

Article Snippet: Spreads were stained with 1x DAPI for 15 min, mounted using Vectashield®, and examined with a Nikon Eclipse TI epifluorescence microscope at 63× magnification using a DAPI filter (Ex 377/50, Em 447/60).

Techniques: Immunostaining, Fluorescence, Flow Cytometry, Expressing, Activity Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Efficient generation of germline chimeras in a non-rodent species using rabbit induced pluripotent stem cells

doi: 10.1038/s41467-025-60314-2

Figure Lengend Snippet: a , b Gel electrophoresis of PCR amplification products for GFP DNA sequences in genomic DNA extracted from peripheral blood cells ( a ) and buccal swabs ( b ) of six viable chimeric animals (one biological replicate). c Confocal microscopy images of tissue sections from chimeras #A2 and #A5 showing GFP + cells labeled with anti-GFP antibody in muscle (scale bar: 250 µm), lung, liver, ovary (50 µm), skin (25 µm), and tongue (500 µm). Images represent three technical replicates per tissue. d Confocal image of a primary ovarian follicle labeled with anti-GFP in a section from female chimera #A6 (scale bar: 75 µm; three technical replicates). e Gel electrophoresis of PCR amplification products for GFP DNA sequences from genomic DNA of E14 F1 embryos obtained after insemination of female chimeras #A2 and #A6 with wild-type sperm (three technical replicates). f Epifluorescence images of whole-mount E14 F1 embryos and a non-chimeric control (one biological replicate). g Identification of four transgene integration sites in KEPi#28 donor cells and F1 fetuses via ligation-mediated PCR. Asterisks (*) indicate specific PCR bands confirmed by sequencing in KEPi#28 and F1 fetuses #16 and #19. Bands observed in other fetuses were not further analyzed. “ns” denotes non-specific PCR products with unreadable sequences (two technical replicates). h PCR amplification of genomic DNA from F1 fetuses confirming the integration of the GFP transgene at a Chromosome 1 locus identified in KEPi#28 cells via ligation-mediated PCR. REF, rabbit embryonic fibroblasts (negative control). Two technical replicates were performed.

Article Snippet: Spreads were stained with 1x DAPI for 15 min, mounted using Vectashield®, and examined with a Nikon Eclipse TI epifluorescence microscope at 63× magnification using a DAPI filter (Ex 377/50, Em 447/60).

Techniques: Nucleic Acid Electrophoresis, Amplification, Confocal Microscopy, Labeling, Control, Ligation, Sequencing, Negative Control

Journal: Journal of Neurochemistry

Article Title: A Computational Approach to Identify Novel Protein Targets Uncovers New Potential Mechanisms of Action of Mirtazapine S (+) and R (−) Enantiomers in Rett Syndrome

doi: 10.1111/jnc.70093

Figure Lengend Snippet: Combined in vitro and in silico workflow for inverse docking target identification. (A) In vitro model to detect drug effects on Mecp2‐KO (RTT) neurons. Hippocampal primary cultures were seeded at DIV 0 in 96 Multi‐Well plates. After 3 days, Ara‐C and drug treatments were administered to the cells. At DIV 6, cells were fixed and immunofluorescence was performed to label dendrites and soma. Images were acquired at the Nikon Eclipse Ti‐E epifluorescence microscope (10x magnification objective) and each image was analyzed with the NeuriteQuant software. (B) Representative images for NeuriteQuant morphological analysis of Total Dendritic Length (TDL) and Endpoints (EPs) of DIV 6 hippocampal Mecp2 ‐KO neurons, plated at the density of 160 cells mm 2 . From left, Mecp2 ‐KO neurons treated with DMSO 0.1% (control condition), Amiloride and Felodipine at the concentration of 10 μM. (C, D) Treatments of neuronal cultures with 10 inactive molecules. Quantitative data of Mecp2 ‐KO neurons, reporting (C) the average TDL per neuron (fold change) and (D) the average number of EPs per neuron. n = 11 images for a total of 1 independent biological replicate (cell cultures). Error bars = mean ± SEM. Red line control condition normalized to = 1 (DMSO 0.1%). One‐way ANOVA (two‐tailed) with Dunnett's multiple comparisons test vs. DMSO conditions. ***p < 0.001, **p < 0.01, *p < 0.05. Grubbs' test was conducted to identify outliers. (E) In silico prediction method. First step: Download of R (−)MTZ and S (+)MTZ chemical structures from PubChem as SDF files; second step: BioGPS screening of R (−)/ S (+) MTZ versus 25 717 human pockets; third step: Comparison of affinity scores versus 10 inactive molecules. A one‐way ANOVA was performed to compare the effects of the ten drugs and the control (DMSO 0.1%). For TDL, F(10, 221) = 4.48, p ‐value = 0.0001. For EP, F(10, 221) = 4.41, p ‐value = 0.0001. Dunnett's multiple comparison test was then used to compare each drug to the control. Adjusted p ‐values for the TDL condition were: Spectinomycin = 0.99, Tizanidine HCl = 0.99, Galanthamine HBr = 0.58, Tubocuranine Cl (+) = 0.98, Alprenolol HCl = 0.99, Meglumine = 0.99, Piroxicam = 0.99, Amilopine = 0.54, Felodipine = 0.58, Pravadoline = 0.98. Adjusted p ‐values for the EP condition were: Spectinomycin = 0.99, Tizanidine HCl = 0.99, Galanthamine HBr = 0.74, Tubocuranine Cl (+) = 0.99, Alprenolol HCl = 0.99, Meglumine = 0.92, Piroxicam = 0.99, Amilopine = 0.38, Felodipine = 0.077, Pravadoline = 0.63.

Article Snippet: Fluorescent images were captured using a Nikon Eclipse Ti‐E epifluorescence live imaging microscope, equipped with a Nikon DS‐Qi2 camera (CMOS sensor, 16.25 megapixel), and a 10X objective lens.

Techniques: In Vitro, In Silico, Drug discovery, Immunofluorescence, Microscopy, Software, Control, Concentration Assay, Two Tailed Test, Comparison