clsm with a 40× objective lens a1 r hd25  (Nikon)

Journal: Scientific Reports

Article Title: Impact of an Alu insertion on the cellular localisation of tissue factor protein

doi: 10.1038/s41598-025-19280-4

Figure Lengend Snippet: Co-transfection of WT and Δ Alu fluorescent constructs. ( a ) The top row shows the WT human F3 3′-UTR construct. For each of the fluorescent constructs the ectodomain has been replaced by either mCherry or mNeonGreen. The Alu element is highlighted by a red box in the 3′-UTR, and red dotted line indicates a deletion of the Alu element; ( b–e ) Fluorescent constructs were co-transfected into MDA-MB-231 cells. Images were acquired as a Z-stack using Plan-Apochromat 63x/1.40 Oil DIC M27 lens on Zeiss LSM-780 inverted confocal microscope. Representative of multiple images, shown as a Z-project. Scale bars, 5 μm. Images analysed on Fiji software (ImageJ). ( b ) mCh-WT and mNG-WT; ( c ) mCh-Δ Alu and mNG-Δ Alu ; ( d ) mNG-WT and mCh-Δ Alu ; and ( e ) mCh-WT and mNG-Δ Alu . Fluorophores encoded by the Δ Alu 3′-UTR F3 constructs localise to the cell surface more readily than those encoded by the WT 3’-UTR F3 constructs.

Article Snippet: Images were acquired as a Z-stack using Plan-Apochromat 63x/1.40 Oil DIC M27 lens on Zeiss LSM-780 inverted confocal microscope.

Techniques: Cotransfection, Construct, Transfection, Microscopy, Software

Journal: Scientific Reports

Article Title: Impact of an Alu insertion on the cellular localisation of tissue factor protein

doi: 10.1038/s41598-025-19280-4

Figure Lengend Snippet: The presence of the Alu element in the F3 3′-UTR affects its localisation within MDA-MB-231 cells. MDA-MB-231 cells were co-transfected with markers to identify specific organelles along with a fluorophore under the influence of the F3 3′-UTR with either the Alu element present (WT) or not (∆ Alu ). ( a ) cell membranes, ( b ) early endosomes, ( c ) Golgi, ( d ) nucleus, ( e ) endoplasmic reticulum. (i) Left columns represent organelle localisation; middle column, WT or Δ Alu F3 ; and the right column, the merged image (colocalisation) of F3 , the organelle marker, and Hoechst 33342 to identify the nucleus (blue). Images were acquired as a Z-stack using Plan-Apochromat 63x/1.40 Oil DIC M27 lens on Zeiss LSM-780 inverted confocal microscope. Representative of multiple images. Scale bars, 5 μm. (ii) Pearson’s correlation coefficient of the colocalisation of the organelle marker with the fluorophore protein under the influence of either the WT or Δ Alu 3′-UTR are shown to the right. The number of cells analysed/the number of independent experiments is indicated on the x axis. Data are expressed as mean ± SEM and analysed using one-way ANOVA. * p < 0.05 ** p < 0.01.

Article Snippet: Images were acquired as a Z-stack using Plan-Apochromat 63x/1.40 Oil DIC M27 lens on Zeiss LSM-780 inverted confocal microscope.

Techniques: Transfection, Marker, Microscopy

Journal: Scientific Reports

Article Title: Impact of an Alu insertion on the cellular localisation of tissue factor protein

doi: 10.1038/s41598-025-19280-4

Figure Lengend Snippet: PAR2-AP increases F3 protein to the cell membrane but has no effect when there is no Alu element present in the F3 3′-UTR. ( a ) Fluorescence confocal microscopy of live MDA-MB-231 cells after transfection of mNG-WT (green, left panels), following stimulation with PAR2-AP (0, 1, 2, or 4 h) and stained with CellMask™ Orange visualising plasma membrane (red), and Hoechst 33,342 (blue). (i) The left panel shows plasma membrane, the middle column shows the fluorescent F3 constructs, and the right panel shows a merged image. Colocalisation of F3 with plasma membrane appears yellow. PAR2-AP increases F3 expression in the cell membrane. Images were acquired as a Z-stack using Plan-Apochromat 63x/1.40 Oil DIC M27 lens on Zeiss LSM-780 inverted confocal microscope. Representative of multiple images. Scale bars, 5 μm. (ii) Pearson’s correlation coefficient of the colocalisation of the plasma membrane with mNeonGreen protein under the influence of F3 ′ s WT 3′-UTR are shown. The number of cells analysed/the number of independent experiments is indicated on the x axis. Data are expressed as mean ± SEM and analysed using one-way ANOVA. * p < 0.05; ( b ) Similar confocal microscopy after transfection of mNG-WT (green) or mNG-Δ Alu (green), following stimulation with PAR2-AP for 4 h and stained with CellMask™ Orange visualising the plasma membrane (red), and DAPI (blue). (i) The left panel shows plasma membrane, the middle column shows the fluorescent F3 constructs, and the right panel shows a merged image. Images were acquired as a Z-stack using Plan-Apochromat 63x/1.40 Oil DIC M27 lens on Zeiss LSM-780 inverted confocal microscope. Representative of multiple images. Scale bars, 5 μm. (ii) Pearson’s correlation coefficient of the colocalisation of the plasma membrane with mNeonGreen protein under the influence of F3 ′ s WT and Δ Alu 3′-UTR are shown. The number of cells analysed/the number of independent experiments is indicated on the x axis. Data are expressed as mean ± SEM and analysed using one-way ANOVA. * p < 0.05.

Article Snippet: Images were acquired as a Z-stack using Plan-Apochromat 63x/1.40 Oil DIC M27 lens on Zeiss LSM-780 inverted confocal microscope.

Techniques: Membrane, Fluorescence, Confocal Microscopy, Transfection, Staining, Clinical Proteomics, Construct, Expressing, Microscopy