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    GE Healthcare cy3
    Fluorescence microscopy of MPO activity. The images show <t>5HT-Cy3</t> staining of MPO – positive cells. 5HT-Cy3 (red fluorescence) detects the presence of active MPO enzyme. ( A ) The infiltration of MPO+ neutrophils from the vascular lumen of human unruptured hBA; ( B ) MPO in cells infiltrated in AVM (magnification – 10x); ( C ) AVM sample showing intracellular and extracellular MPO at high magnification (40x), L- vascular lumen; ( D ) high-magnification image of infiltrating neutrophils in unruptured hBA (see panel A) by using MPO and anti-calprotectin (S100A8/A9 complex) detection (63x); ( E ) - high-magnification image of infiltrating neutrophils in AVM (see panel C) by using MPO and calprotectin detection. Red- 5HT-Cy3, green - anti-calprotectin antibody, blue - DAPI. Bars ( D,E ) = 25 µm.
    Cy3, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy3/product/GE Healthcare
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cy3 - by Bioz Stars, 2021-04
    86/100 stars
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    99
    Jackson Immuno cy3
    Fluorescence microscopy of MPO activity. The images show <t>5HT-Cy3</t> staining of MPO – positive cells. 5HT-Cy3 (red fluorescence) detects the presence of active MPO enzyme. ( A ) The infiltration of MPO+ neutrophils from the vascular lumen of human unruptured hBA; ( B ) MPO in cells infiltrated in AVM (magnification – 10x); ( C ) AVM sample showing intracellular and extracellular MPO at high magnification (40x), L- vascular lumen; ( D ) high-magnification image of infiltrating neutrophils in unruptured hBA (see panel A) by using MPO and anti-calprotectin (S100A8/A9 complex) detection (63x); ( E ) - high-magnification image of infiltrating neutrophils in AVM (see panel C) by using MPO and calprotectin detection. Red- 5HT-Cy3, green - anti-calprotectin antibody, blue - DAPI. Bars ( D,E ) = 25 µm.
    Cy3, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy3/product/Jackson Immuno
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cy3 - by Bioz Stars, 2021-04
    99/100 stars
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    86
    Agilent technologies cy3
    Fig. 8. Telomeric DNA associates with PML bodies in a dynamic manner. U2OS cells were transiently transfected with CFP–PML ( A ) and loaded with <t>cy3-labeled</t> (C 3 TA 2 ) 3 PNA ( B ). A subset of telomeres were shown to be associated with PML bodies ( C ). A differential contrast image of the cell in (A)–(C) is shown in ( D ). The colocalization of telomeric DNA with PML in this cell is more clearly shown in a mask image ( E ) that was obtained by selecting those pixels in which the intensity of both colors was above a threshold value that distinguished the specific signals from background fluorescence ( F ). Time-lapse images of another cell nucleus, taken every 90 s for 30 min, reveal successively the association of a telomere with a PML body, its dissociation from the body and its association with another PML body ( G ). Occasionally, PML protein was shown to localize to a telomeric DNA sequence before their association with a PML nuclear body ( H ). In the lower row, the colocalization between telomeric DNA and PML is shown in white by selecting pixels in which the intensity of both colors is above threshold value.
    Cy3, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy3/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cy3 - by Bioz Stars, 2021-04
    86/100 stars
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    86
    Jackson Immuno cy3 conjugated secondary antibodies
    BiFC analysis confirms that B56γ3, but not B55α, promotes PP2A/Aα accumulation in the nucleus in early S phase. ( A ) NIH3T3 cells stably expressing HA-tagged B56γ3 or B55α were synchronized at the early S phase by double thymidine block treatment [48] followed by released in the regular medium for 3 h as described before [29] . Expression of B56γ3 or B55α was assessed by indirect immunofluorescence using anti-HA antibody in conjunction with <t>Cy3-conjugated</t> secondary antibody. ( B ) Equal amounts of BiFC expression constructs encoding YC-Aα and YN-B56γ3 or equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α were co-transfected into NIH3T3 cells. Twenty-four hour after transfection, cells were either treated with 10 µg/ml aphidicolin or left untreated for 18 h and subsequently grown in fresh medium without aphidicolin treatment for 3 h, followed by direct fluorescence microscopy for imaging YFP signals due to BiFC of YC-Aα and YN-B56γ3 or BiFC of Aα-YC and YN-B55α. DAPI was applied for staining of nuclei. Scale bars, 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.
    Cy3 Conjugated Secondary Antibodies, supplied by Jackson Immuno, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cy3 conjugated secondary antibodies/product/Jackson Immuno
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cy3 conjugated secondary antibodies - by Bioz Stars, 2021-04
    86/100 stars
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    ChromPure is our trade name for highly purified proteins from the serum of non immunized animals
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    N/A
    ChromPure is our trade name for highly purified proteins from the serum of non immunized animals
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    N/A
    ChromPure is our trade name for highly purified proteins from the serum of non immunized animals
      Buy from Supplier

    N/A
    ChromPure is our trade name for highly purified proteins from the serum of non immunized animals
      Buy from Supplier

    Image Search Results


    Fluorescence microscopy of MPO activity. The images show 5HT-Cy3 staining of MPO – positive cells. 5HT-Cy3 (red fluorescence) detects the presence of active MPO enzyme. ( A ) The infiltration of MPO+ neutrophils from the vascular lumen of human unruptured hBA; ( B ) MPO in cells infiltrated in AVM (magnification – 10x); ( C ) AVM sample showing intracellular and extracellular MPO at high magnification (40x), L- vascular lumen; ( D ) high-magnification image of infiltrating neutrophils in unruptured hBA (see panel A) by using MPO and anti-calprotectin (S100A8/A9 complex) detection (63x); ( E ) - high-magnification image of infiltrating neutrophils in AVM (see panel C) by using MPO and calprotectin detection. Red- 5HT-Cy3, green - anti-calprotectin antibody, blue - DAPI. Bars ( D,E ) = 25 µm.

    Journal: Scientific Reports

    Article Title: High-resolution Imaging of Myeloperoxidase Activity Sensors in Human Cerebrovascular Disease

    doi: 10.1038/s41598-018-25804-y

    Figure Lengend Snippet: Fluorescence microscopy of MPO activity. The images show 5HT-Cy3 staining of MPO – positive cells. 5HT-Cy3 (red fluorescence) detects the presence of active MPO enzyme. ( A ) The infiltration of MPO+ neutrophils from the vascular lumen of human unruptured hBA; ( B ) MPO in cells infiltrated in AVM (magnification – 10x); ( C ) AVM sample showing intracellular and extracellular MPO at high magnification (40x), L- vascular lumen; ( D ) high-magnification image of infiltrating neutrophils in unruptured hBA (see panel A) by using MPO and anti-calprotectin (S100A8/A9 complex) detection (63x); ( E ) - high-magnification image of infiltrating neutrophils in AVM (see panel C) by using MPO and calprotectin detection. Red- 5HT-Cy3, green - anti-calprotectin antibody, blue - DAPI. Bars ( D,E ) = 25 µm.

    Article Snippet: Briefly, 5HT-Cy3 was synthesized by reacting 1.1 molar excess of 5-hydroxytryptamine with mono N-hydroxysuccinimide ester of Cy3 (GE Healthcare Bios-Sciences Corp., Piscataway NJ) in DMF in the presence of triethylamine under argon.

    Techniques: Fluorescence, Microscopy, Activity Assay, Staining

    Comparison of µMRI of large unruptured hBA with the results of histology and fluorescence microscopy. ( A ) MRI (UTE pulse sequence) of a thick section of hBA treated with Gd-bis-5HT-DTPA and imaged by using µMRI coil; ( B ) trichrome stain of a consecutive thin section, magnification -10x; C- fluorescence microscopy of a thick section stained by using fluorescent MPO reducing substrate 5HT-Cy3, magnification -4x. Scale bar = 1 mm. Arrowheads point to the area of thrombus containing infiltrated neutrophils and MPO. Asterisks indicate the location of adventitial vessel (evidence of adventitial angiogenesis), containing MPO-positive cells.

    Journal: Scientific Reports

    Article Title: High-resolution Imaging of Myeloperoxidase Activity Sensors in Human Cerebrovascular Disease

    doi: 10.1038/s41598-018-25804-y

    Figure Lengend Snippet: Comparison of µMRI of large unruptured hBA with the results of histology and fluorescence microscopy. ( A ) MRI (UTE pulse sequence) of a thick section of hBA treated with Gd-bis-5HT-DTPA and imaged by using µMRI coil; ( B ) trichrome stain of a consecutive thin section, magnification -10x; C- fluorescence microscopy of a thick section stained by using fluorescent MPO reducing substrate 5HT-Cy3, magnification -4x. Scale bar = 1 mm. Arrowheads point to the area of thrombus containing infiltrated neutrophils and MPO. Asterisks indicate the location of adventitial vessel (evidence of adventitial angiogenesis), containing MPO-positive cells.

    Article Snippet: Briefly, 5HT-Cy3 was synthesized by reacting 1.1 molar excess of 5-hydroxytryptamine with mono N-hydroxysuccinimide ester of Cy3 (GE Healthcare Bios-Sciences Corp., Piscataway NJ) in DMF in the presence of triethylamine under argon.

    Techniques: Fluorescence, Microscopy, Magnetic Resonance Imaging, Sequencing, Staining

    Fluorescence and 7T µMRI imaging of a tissue-like phantom. Phantom consisted of 5 wells containing thick (100 µm) frozen sections of tissue-like material containing various specific enzymatic activity of MPO (well 1 = 0, well 2 = 0.1, well 3 = 0.2, well 4 = 0.6 and well 5 = 1.0 U MPO/µl). MPO was dissolved and embedded in agarose/acrylamide/Matrigel mixture. ( A ) Phantom appearance, the colour is due to phenol red in the Matrigel samples 1–5 immersed in OCT medium; ( B ) fluorescence microscopy of thin sections of the phantom in A treated with 0.1 mM 5HT-Cy3/1 mM H 2 O 2 after removing of unreacted 5HT-Cy3; ( C ) µMR imaging of the phantom after incubating 0.1 mm-thick sections in the presence and 0.5 mM bis-5HT-Gd-DTPA/1 mM H 2 O 2 (UTE pulse sequence acquisition, with image inversion); ( D ) Relaxation rates R1 and R2 (measured by using µMRI of thick sections) as a function of MPO concentration in the phantom components. Data shown as mean ± SD (n = 3).

    Journal: Scientific Reports

    Article Title: High-resolution Imaging of Myeloperoxidase Activity Sensors in Human Cerebrovascular Disease

    doi: 10.1038/s41598-018-25804-y

    Figure Lengend Snippet: Fluorescence and 7T µMRI imaging of a tissue-like phantom. Phantom consisted of 5 wells containing thick (100 µm) frozen sections of tissue-like material containing various specific enzymatic activity of MPO (well 1 = 0, well 2 = 0.1, well 3 = 0.2, well 4 = 0.6 and well 5 = 1.0 U MPO/µl). MPO was dissolved and embedded in agarose/acrylamide/Matrigel mixture. ( A ) Phantom appearance, the colour is due to phenol red in the Matrigel samples 1–5 immersed in OCT medium; ( B ) fluorescence microscopy of thin sections of the phantom in A treated with 0.1 mM 5HT-Cy3/1 mM H 2 O 2 after removing of unreacted 5HT-Cy3; ( C ) µMR imaging of the phantom after incubating 0.1 mm-thick sections in the presence and 0.5 mM bis-5HT-Gd-DTPA/1 mM H 2 O 2 (UTE pulse sequence acquisition, with image inversion); ( D ) Relaxation rates R1 and R2 (measured by using µMRI of thick sections) as a function of MPO concentration in the phantom components. Data shown as mean ± SD (n = 3).

    Article Snippet: Briefly, 5HT-Cy3 was synthesized by reacting 1.1 molar excess of 5-hydroxytryptamine with mono N-hydroxysuccinimide ester of Cy3 (GE Healthcare Bios-Sciences Corp., Piscataway NJ) in DMF in the presence of triethylamine under argon.

    Techniques: Fluorescence, Imaging, Activity Assay, Microscopy, Sequencing, Concentration Assay

    µMR Imaging and correlative immunohistochemistry of human arteriovenous malformation and unruptured human aneurysm. ( A ) µMRI image of a thick section (80 µm) of the arteriovenous malformation (AVM) showing moderate to strong T1-weighted signal detected over the circumference of the structures 3 and 4; ( B ) matching section showing immunohistochemical (IHC) detection of MPO (blue); ( C ) µMRI image of a left middle cerebral artery unruptured human aneurysm tissue sample; ( D ) a light microscopy image of thick section shown in panel C; ( E ) an anti-MPO IHC image (thin section). MPO positive cells (blue, 20x magnification) are pointed to by an arrow (inset). ( F ) Red fluorescence of MPO+ cells and extracellular MPO. Anti-MPO staining was performed by treating the tissue sequentially with anti-MPO mAb (anti-human mouse monoclonal antibody (1:200, AbCam), followed by a secondary mAb-alkaline phosphatase conjugate (1:500, Roche.). Yellow arrow is pointing to the area matched to the inset of panel (E, black arrow). This area is positive by MPO immunofluorescence ( E ) and MPO activity ( F ). Bar = 50 µm. In B and C the counterstaining of nuclei was performed by using Nuclear Fast Red (Vector labs) and DAPI was used for image F. MPO activity was revealed by incubating the section in the presence of 0.1 mM 5HT-Cy3/1 mM H 2 O 2 for 30 min.

    Journal: Scientific Reports

    Article Title: High-resolution Imaging of Myeloperoxidase Activity Sensors in Human Cerebrovascular Disease

    doi: 10.1038/s41598-018-25804-y

    Figure Lengend Snippet: µMR Imaging and correlative immunohistochemistry of human arteriovenous malformation and unruptured human aneurysm. ( A ) µMRI image of a thick section (80 µm) of the arteriovenous malformation (AVM) showing moderate to strong T1-weighted signal detected over the circumference of the structures 3 and 4; ( B ) matching section showing immunohistochemical (IHC) detection of MPO (blue); ( C ) µMRI image of a left middle cerebral artery unruptured human aneurysm tissue sample; ( D ) a light microscopy image of thick section shown in panel C; ( E ) an anti-MPO IHC image (thin section). MPO positive cells (blue, 20x magnification) are pointed to by an arrow (inset). ( F ) Red fluorescence of MPO+ cells and extracellular MPO. Anti-MPO staining was performed by treating the tissue sequentially with anti-MPO mAb (anti-human mouse monoclonal antibody (1:200, AbCam), followed by a secondary mAb-alkaline phosphatase conjugate (1:500, Roche.). Yellow arrow is pointing to the area matched to the inset of panel (E, black arrow). This area is positive by MPO immunofluorescence ( E ) and MPO activity ( F ). Bar = 50 µm. In B and C the counterstaining of nuclei was performed by using Nuclear Fast Red (Vector labs) and DAPI was used for image F. MPO activity was revealed by incubating the section in the presence of 0.1 mM 5HT-Cy3/1 mM H 2 O 2 for 30 min.

    Article Snippet: Briefly, 5HT-Cy3 was synthesized by reacting 1.1 molar excess of 5-hydroxytryptamine with mono N-hydroxysuccinimide ester of Cy3 (GE Healthcare Bios-Sciences Corp., Piscataway NJ) in DMF in the presence of triethylamine under argon.

    Techniques: Imaging, Immunohistochemistry, Light Microscopy, Fluorescence, Staining, Immunofluorescence, Activity Assay, Plasmid Preparation

    Fig. 8. Telomeric DNA associates with PML bodies in a dynamic manner. U2OS cells were transiently transfected with CFP–PML ( A ) and loaded with cy3-labeled (C 3 TA 2 ) 3 PNA ( B ). A subset of telomeres were shown to be associated with PML bodies ( C ). A differential contrast image of the cell in (A)–(C) is shown in ( D ). The colocalization of telomeric DNA with PML in this cell is more clearly shown in a mask image ( E ) that was obtained by selecting those pixels in which the intensity of both colors was above a threshold value that distinguished the specific signals from background fluorescence ( F ). Time-lapse images of another cell nucleus, taken every 90 s for 30 min, reveal successively the association of a telomere with a PML body, its dissociation from the body and its association with another PML body ( G ). Occasionally, PML protein was shown to localize to a telomeric DNA sequence before their association with a PML nuclear body ( H ). In the lower row, the colocalization between telomeric DNA and PML is shown in white by selecting pixels in which the intensity of both colors is above threshold value.

    Journal: The EMBO Journal

    Article Title: Visualizing telomere dynamics in living mammalian cells using PNA probes

    doi: 10.1093/emboj/cdg633

    Figure Lengend Snippet: Fig. 8. Telomeric DNA associates with PML bodies in a dynamic manner. U2OS cells were transiently transfected with CFP–PML ( A ) and loaded with cy3-labeled (C 3 TA 2 ) 3 PNA ( B ). A subset of telomeres were shown to be associated with PML bodies ( C ). A differential contrast image of the cell in (A)–(C) is shown in ( D ). The colocalization of telomeric DNA with PML in this cell is more clearly shown in a mask image ( E ) that was obtained by selecting those pixels in which the intensity of both colors was above a threshold value that distinguished the specific signals from background fluorescence ( F ). Time-lapse images of another cell nucleus, taken every 90 s for 30 min, reveal successively the association of a telomere with a PML body, its dissociation from the body and its association with another PML body ( G ). Occasionally, PML protein was shown to localize to a telomeric DNA sequence before their association with a PML nuclear body ( H ). In the lower row, the colocalization between telomeric DNA and PML is shown in white by selecting pixels in which the intensity of both colors is above threshold value.

    Article Snippet: Cy3- and lissamine-labeled (C3 TA2 )3 PNA probes (DAKO) were dissolved in a buffer containing 80 mM KCl, 10 mM K2 PO4 , 4 mM NaCl, pH 7.2, to a final concentration of 1 µM.

    Techniques: Transfection, Labeling, Fluorescence, Sequencing

    Fig. 5. Time-lapse microscopy demonstrates temporal interactions between telomeric DNA foci. U2OS cells were loaded with cy3-labeled (C 3 TA 2 ) 3 PNA and image stacks were recorded at 60-s time-intervals for 1 h. In the cell shown in ( A – H ), two independent fusion events were recorded as indicated by arrowheads. In another interphase cell nucleus, the separation of two telomeric DNA foci is shown ( J – L , arrows). Images represent maximum projections of 16 Z-sections.

    Journal: The EMBO Journal

    Article Title: Visualizing telomere dynamics in living mammalian cells using PNA probes

    doi: 10.1093/emboj/cdg633

    Figure Lengend Snippet: Fig. 5. Time-lapse microscopy demonstrates temporal interactions between telomeric DNA foci. U2OS cells were loaded with cy3-labeled (C 3 TA 2 ) 3 PNA and image stacks were recorded at 60-s time-intervals for 1 h. In the cell shown in ( A – H ), two independent fusion events were recorded as indicated by arrowheads. In another interphase cell nucleus, the separation of two telomeric DNA foci is shown ( J – L , arrows). Images represent maximum projections of 16 Z-sections.

    Article Snippet: Cy3- and lissamine-labeled (C3 TA2 )3 PNA probes (DAKO) were dissolved in a buffer containing 80 mM KCl, 10 mM K2 PO4 , 4 mM NaCl, pH 7.2, to a final concentration of 1 µM.

    Techniques: Time-lapse Microscopy, Labeling

    Fig. 4. Quantitative analysis of telomere movements. ( A ) A plot of MSD against Δ t for telomeres stained with either cy3-PNA or CFP–TRF2. Data represent average values of 100 telomeres (derived from five cells) for cy3-PNA and 25 telomeres for CFP–TRF2 (derived from two cells). Both plots show an indicative for constraint diffusion and reach a plateau at ∼0.2 µm 2 /s. ( B ) MSD plot of three populations of cy3-PNA labeled telomeres demonstrating different movement behavior. The squares represent the most constrained population, which is also shown in (A). The triangles represent a subpopulation of telomeres (10% of all telomeres) that show faster movement over larger distances but that still reach a plateau. The diamonds represent a fast-moving telomere. By plotting MSD/Δ t , proportional to the diffusion coefficent, as a function of Δ t ), it becomes clear that the diffusion coefficient decreases with increasing time intervals for the two slowest populations ( C and D ), consistent with constrained movement, but not for the fast telomere for the time period analyzed (D).

    Journal: The EMBO Journal

    Article Title: Visualizing telomere dynamics in living mammalian cells using PNA probes

    doi: 10.1093/emboj/cdg633

    Figure Lengend Snippet: Fig. 4. Quantitative analysis of telomere movements. ( A ) A plot of MSD against Δ t for telomeres stained with either cy3-PNA or CFP–TRF2. Data represent average values of 100 telomeres (derived from five cells) for cy3-PNA and 25 telomeres for CFP–TRF2 (derived from two cells). Both plots show an indicative for constraint diffusion and reach a plateau at ∼0.2 µm 2 /s. ( B ) MSD plot of three populations of cy3-PNA labeled telomeres demonstrating different movement behavior. The squares represent the most constrained population, which is also shown in (A). The triangles represent a subpopulation of telomeres (10% of all telomeres) that show faster movement over larger distances but that still reach a plateau. The diamonds represent a fast-moving telomere. By plotting MSD/Δ t , proportional to the diffusion coefficent, as a function of Δ t ), it becomes clear that the diffusion coefficient decreases with increasing time intervals for the two slowest populations ( C and D ), consistent with constrained movement, but not for the fast telomere for the time period analyzed (D).

    Article Snippet: Cy3- and lissamine-labeled (C3 TA2 )3 PNA probes (DAKO) were dissolved in a buffer containing 80 mM KCl, 10 mM K2 PO4 , 4 mM NaCl, pH 7.2, to a final concentration of 1 µM.

    Techniques: Staining, Derivative Assay, Diffusion-based Assay, Labeling

    Fig. 1. Telomere-specific PNA probe binds to telomeric DNA sequences. Human U2OS as well as mouse MS5 cells were bead-loaded with a cy3-labeled (C 3 TA 2 ) 3 PNA probe and monitored 2 h later. Binding of the probe to telomeric DNA in U2OS cells resulted in variable numbers of fluorescent spots that varied in size and intensity ( A and B ). The image of the cell depicted in (A) reveals 54 spots, while in the two cells shown in (B), 51 and 65 spots are visible. In addition to interphase cells, binding of PNA probe to telomeres in mitotic cells was also observed as indicated by the differential interference contrast image ( C ) and the fluorescence image ( D ). In mouse MS5 cells, telomeres localized to two heterochromatic regions, which are indicated by arrows in the phase contrast image ( E ) in addition to other nuclear sites ( F ). Living U2OS cells that were first loaded with lissamine- labeled PNA probe ( G ), and then fixed and hybridized with a FITC- labeled, telomere-specific plasmid probe ( H ), show colocalization as indicated in yellow in ( I ). Hybridization of cy3-labeled (C 3 TA 2 ) 3 PNA probe to metaphase spreads of U2OS cells revealed staining of telomeric DNA at nearly all chromosome ends, in addition to staining of a few extrachromosomal telomeric DNA repeats ( K and L ). Chromosomes were counterstained with DAPI ( J ). Bar = 5 µm.

    Journal: The EMBO Journal

    Article Title: Visualizing telomere dynamics in living mammalian cells using PNA probes

    doi: 10.1093/emboj/cdg633

    Figure Lengend Snippet: Fig. 1. Telomere-specific PNA probe binds to telomeric DNA sequences. Human U2OS as well as mouse MS5 cells were bead-loaded with a cy3-labeled (C 3 TA 2 ) 3 PNA probe and monitored 2 h later. Binding of the probe to telomeric DNA in U2OS cells resulted in variable numbers of fluorescent spots that varied in size and intensity ( A and B ). The image of the cell depicted in (A) reveals 54 spots, while in the two cells shown in (B), 51 and 65 spots are visible. In addition to interphase cells, binding of PNA probe to telomeres in mitotic cells was also observed as indicated by the differential interference contrast image ( C ) and the fluorescence image ( D ). In mouse MS5 cells, telomeres localized to two heterochromatic regions, which are indicated by arrows in the phase contrast image ( E ) in addition to other nuclear sites ( F ). Living U2OS cells that were first loaded with lissamine- labeled PNA probe ( G ), and then fixed and hybridized with a FITC- labeled, telomere-specific plasmid probe ( H ), show colocalization as indicated in yellow in ( I ). Hybridization of cy3-labeled (C 3 TA 2 ) 3 PNA probe to metaphase spreads of U2OS cells revealed staining of telomeric DNA at nearly all chromosome ends, in addition to staining of a few extrachromosomal telomeric DNA repeats ( K and L ). Chromosomes were counterstained with DAPI ( J ). Bar = 5 µm.

    Article Snippet: Cy3- and lissamine-labeled (C3 TA2 )3 PNA probes (DAKO) were dissolved in a buffer containing 80 mM KCl, 10 mM K2 PO4 , 4 mM NaCl, pH 7.2, to a final concentration of 1 µM.

    Techniques: Labeling, Binding Assay, Fluorescence, Plasmid Preparation, Hybridization, Staining

    Fig. 3. Live-cell imaging demonstrates dynamics of telomeric DNA. U2OS cells were loaded with cy3-labeled (C 3 TA 2 ) 3 PNA and image stacks were collected every 90 s for 60 min. During two different time periods, the movements of telomeres [indicated by arrows and arrowheads in ( A )] were recorded and the trajectories were plotted ( B and C ). As indicated by the length of the trajectories, some telomeres showed a significant movement while others showed hardly any movement. ( D ) Another example of a cell nucleus in which a telomere spot (arrowhead) moved over a large distance within a defined (6 min) time period. From the cell in (A)–(C), the trajectories of a selected number of telomeres in the first 20 min of the time-lapse series are calculated and displayed in the xy -plane ( E ). While most telomeres moved in a small area, the spot indicated by trajectory number 13 showed significant movement. This difference in mobility becomes more apparent when the trajectories ( F ) and velocities ( G ) of spots 10 (green) and 13 (purple) are compared. In (F), the trajectory of the two telomeres is shown from two different viewpoints.

    Journal: The EMBO Journal

    Article Title: Visualizing telomere dynamics in living mammalian cells using PNA probes

    doi: 10.1093/emboj/cdg633

    Figure Lengend Snippet: Fig. 3. Live-cell imaging demonstrates dynamics of telomeric DNA. U2OS cells were loaded with cy3-labeled (C 3 TA 2 ) 3 PNA and image stacks were collected every 90 s for 60 min. During two different time periods, the movements of telomeres [indicated by arrows and arrowheads in ( A )] were recorded and the trajectories were plotted ( B and C ). As indicated by the length of the trajectories, some telomeres showed a significant movement while others showed hardly any movement. ( D ) Another example of a cell nucleus in which a telomere spot (arrowhead) moved over a large distance within a defined (6 min) time period. From the cell in (A)–(C), the trajectories of a selected number of telomeres in the first 20 min of the time-lapse series are calculated and displayed in the xy -plane ( E ). While most telomeres moved in a small area, the spot indicated by trajectory number 13 showed significant movement. This difference in mobility becomes more apparent when the trajectories ( F ) and velocities ( G ) of spots 10 (green) and 13 (purple) are compared. In (F), the trajectory of the two telomeres is shown from two different viewpoints.

    Article Snippet: Cy3- and lissamine-labeled (C3 TA2 )3 PNA probes (DAKO) were dissolved in a buffer containing 80 mM KCl, 10 mM K2 PO4 , 4 mM NaCl, pH 7.2, to a final concentration of 1 µM.

    Techniques: Live Cell Imaging, Labeling

    Fig. 2. Hybridization of PNAs to telomeric DNA does not prevent binding of CFP–TRF2 in living U2OS cells. Double labeling of cells with CFP–TRF2 and cy3-PNA results in a nearly complete colocalization of the two (C). Hybridization of cy3-PNA to telomeres is shown in ( A ) and recruitment of CFP–TRF2 to telomeres is shown in ( B ). ( C ) A few telomeres, indicated with arrowheads, were stained with either PNA probe or CFP–TRF2 only. A differential interference contrast image of this nucleus is shown in ( D ).

    Journal: The EMBO Journal

    Article Title: Visualizing telomere dynamics in living mammalian cells using PNA probes

    doi: 10.1093/emboj/cdg633

    Figure Lengend Snippet: Fig. 2. Hybridization of PNAs to telomeric DNA does not prevent binding of CFP–TRF2 in living U2OS cells. Double labeling of cells with CFP–TRF2 and cy3-PNA results in a nearly complete colocalization of the two (C). Hybridization of cy3-PNA to telomeres is shown in ( A ) and recruitment of CFP–TRF2 to telomeres is shown in ( B ). ( C ) A few telomeres, indicated with arrowheads, were stained with either PNA probe or CFP–TRF2 only. A differential interference contrast image of this nucleus is shown in ( D ).

    Article Snippet: Cy3- and lissamine-labeled (C3 TA2 )3 PNA probes (DAKO) were dissolved in a buffer containing 80 mM KCl, 10 mM K2 PO4 , 4 mM NaCl, pH 7.2, to a final concentration of 1 µM.

    Techniques: Hybridization, Binding Assay, Labeling, Staining

    BiFC analysis confirms that B56γ3, but not B55α, promotes PP2A/Aα accumulation in the nucleus in early S phase. ( A ) NIH3T3 cells stably expressing HA-tagged B56γ3 or B55α were synchronized at the early S phase by double thymidine block treatment [48] followed by released in the regular medium for 3 h as described before [29] . Expression of B56γ3 or B55α was assessed by indirect immunofluorescence using anti-HA antibody in conjunction with Cy3-conjugated secondary antibody. ( B ) Equal amounts of BiFC expression constructs encoding YC-Aα and YN-B56γ3 or equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α were co-transfected into NIH3T3 cells. Twenty-four hour after transfection, cells were either treated with 10 µg/ml aphidicolin or left untreated for 18 h and subsequently grown in fresh medium without aphidicolin treatment for 3 h, followed by direct fluorescence microscopy for imaging YFP signals due to BiFC of YC-Aα and YN-B56γ3 or BiFC of Aα-YC and YN-B55α. DAPI was applied for staining of nuclei. Scale bars, 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.

    Journal: PLoS ONE

    Article Title: Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

    doi: 10.1371/journal.pone.0116074

    Figure Lengend Snippet: BiFC analysis confirms that B56γ3, but not B55α, promotes PP2A/Aα accumulation in the nucleus in early S phase. ( A ) NIH3T3 cells stably expressing HA-tagged B56γ3 or B55α were synchronized at the early S phase by double thymidine block treatment [48] followed by released in the regular medium for 3 h as described before [29] . Expression of B56γ3 or B55α was assessed by indirect immunofluorescence using anti-HA antibody in conjunction with Cy3-conjugated secondary antibody. ( B ) Equal amounts of BiFC expression constructs encoding YC-Aα and YN-B56γ3 or equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α were co-transfected into NIH3T3 cells. Twenty-four hour after transfection, cells were either treated with 10 µg/ml aphidicolin or left untreated for 18 h and subsequently grown in fresh medium without aphidicolin treatment for 3 h, followed by direct fluorescence microscopy for imaging YFP signals due to BiFC of YC-Aα and YN-B56γ3 or BiFC of Aα-YC and YN-B55α. DAPI was applied for staining of nuclei. Scale bars, 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.

    Article Snippet: Cells were then blocked with 5% BSA in PBS for 1 h, and subsequently incubated with anti-HA (Cell Signaling, 2367) or anti-Myc tag (Cell Signaling, 2278) antibody for 1 h, followed by incubation with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Bimolecular Fluorescence Complementation Assay, Stable Transfection, Expressing, Blocking Assay, Immunofluorescence, Construct, Transfection, Fluorescence, Microscopy, Imaging, Staining

    Visualization of the α4/PP2Ac complex by BiFC. ( A ) BiFC expression constructs encoding YN-α4 WT or YN-α4 MUT and YC-PP2Acα were transfected into NIH3T3 cells. YFP signals due to BiFC of YN-α4 WT or BiFC of YN-α4 MUT and YC-PP2Acα were measured by fluorescence microscopy. Expression of Myc-tagged YN-α4 WT or YN-α4 MUT was confirmed using anti-Myc antibody in conjunction with Cy3-conjugated secondary antibody by indirect immunofluorescence microscopy. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as described earlier. Quantified data from one of at least two independent experiments with similar results are shown. At least 150 cells were counted for each group. ( B ) BiFC expression constructs encoding YN-α4 WT or YN-α4 MUT and YC-PP2Acα were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection and immunoprecipitations were performed using anti-Myc tag antibody. The cell lysates and anti-Myc tag immunocomplexes were then analyzed by SDS-PAGE and Western blotting using the indicated antibodies.

    Journal: PLoS ONE

    Article Title: Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

    doi: 10.1371/journal.pone.0116074

    Figure Lengend Snippet: Visualization of the α4/PP2Ac complex by BiFC. ( A ) BiFC expression constructs encoding YN-α4 WT or YN-α4 MUT and YC-PP2Acα were transfected into NIH3T3 cells. YFP signals due to BiFC of YN-α4 WT or BiFC of YN-α4 MUT and YC-PP2Acα were measured by fluorescence microscopy. Expression of Myc-tagged YN-α4 WT or YN-α4 MUT was confirmed using anti-Myc antibody in conjunction with Cy3-conjugated secondary antibody by indirect immunofluorescence microscopy. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as described earlier. Quantified data from one of at least two independent experiments with similar results are shown. At least 150 cells were counted for each group. ( B ) BiFC expression constructs encoding YN-α4 WT or YN-α4 MUT and YC-PP2Acα were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection and immunoprecipitations were performed using anti-Myc tag antibody. The cell lysates and anti-Myc tag immunocomplexes were then analyzed by SDS-PAGE and Western blotting using the indicated antibodies.

    Article Snippet: Cells were then blocked with 5% BSA in PBS for 1 h, and subsequently incubated with anti-HA (Cell Signaling, 2367) or anti-Myc tag (Cell Signaling, 2278) antibody for 1 h, followed by incubation with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Bimolecular Fluorescence Complementation Assay, Expressing, Construct, Transfection, Fluorescence, Microscopy, Immunofluorescence, Staining, SDS Page, Western Blot

    BiFC analysis enables visualization of association between two subunits of PP2A in cells. ( A ) Design of BiFC analysis of dimeric interactions between PP2A subunits is shown. Fluorescence is regained when reconstitution of YFP from two fragments of YFP takes place due to an interaction between PP2A subunits fused to the fragments. ( B ) Equal amounts of BiFC expression constructs encoding YN-Aα and PP2Acα-YC were co-transfected into NIH3T3 cells. YFP signals due to BiFC of YN-Aα and PP2Acα-YC were measured by fluorescence microscopy. ( C ) Equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α, YN-B55β1, YN-B55β2, YN-B55βαβ, or YN-B55δ, or constructs encoding YC-Aα and YN-B56γ3 were transfected into NIH3T3 cells. YFP signals due to BiFC of Aα-YC and YN-B were measured by fluorescence microscopy. ( D ) Equal amounts of BiFC expression constructs encoding PP2Acα-YC and YN-B55β1 or YN-B56γ3 with or without equal amounts of pCA2-6myc-PP2A/Aα were co-transfected into NIH3T3 cells, and 24 h after transfection, YFP signals due to BiFC of PP2Acα-YC and YN-B55β1 or YN-B56γ3 were measured by direct fluorescence microscopy and expression of 6myc-PP2A/Aα was confirmed by indirect immunofluorescence using anti-Myc tag antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.

    Journal: PLoS ONE

    Article Title: Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

    doi: 10.1371/journal.pone.0116074

    Figure Lengend Snippet: BiFC analysis enables visualization of association between two subunits of PP2A in cells. ( A ) Design of BiFC analysis of dimeric interactions between PP2A subunits is shown. Fluorescence is regained when reconstitution of YFP from two fragments of YFP takes place due to an interaction between PP2A subunits fused to the fragments. ( B ) Equal amounts of BiFC expression constructs encoding YN-Aα and PP2Acα-YC were co-transfected into NIH3T3 cells. YFP signals due to BiFC of YN-Aα and PP2Acα-YC were measured by fluorescence microscopy. ( C ) Equal amounts of BiFC expression constructs encoding Aα-YC and YN-B55α, YN-B55β1, YN-B55β2, YN-B55βαβ, or YN-B55δ, or constructs encoding YC-Aα and YN-B56γ3 were transfected into NIH3T3 cells. YFP signals due to BiFC of Aα-YC and YN-B were measured by fluorescence microscopy. ( D ) Equal amounts of BiFC expression constructs encoding PP2Acα-YC and YN-B55β1 or YN-B56γ3 with or without equal amounts of pCA2-6myc-PP2A/Aα were co-transfected into NIH3T3 cells, and 24 h after transfection, YFP signals due to BiFC of PP2Acα-YC and YN-B55β1 or YN-B56γ3 were measured by direct fluorescence microscopy and expression of 6myc-PP2A/Aα was confirmed by indirect immunofluorescence using anti-Myc tag antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Graphs show quantitative analysis of distribution of BiFC signals in cells from one of at least two independent experiments with similar results, and at least 100 cells were assessed from several random fields. Cells with different distribution patterns of BiFC signals were scored as described earlier.

    Article Snippet: Cells were then blocked with 5% BSA in PBS for 1 h, and subsequently incubated with anti-HA (Cell Signaling, 2367) or anti-Myc tag (Cell Signaling, 2278) antibody for 1 h, followed by incubation with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, Expressing, Construct, Transfection, Microscopy, Immunofluorescence, Staining

    Subcellular distribution of PP2A subunits. ( A ) NIH3T3 cells were transiently transfected with pCA2-6myc-PP2A/Aα or pCMV-HA-PP2Acα-YC, and expression of the exogenous Aα and Cα subunits was assessed by indirect immunofluorescence using anti-Myc tag and anti-HA antibodies, respectively, in conjunction with Cy3-conjugated secondary antibody. ( B ) Diagrams of B55α, B55β, and the B55βαβ chimera mutant are shown. ( C ) NIH3T3 cells were transiently transfected with pcDNA3.1/Zeo(+)-B55α-HA, pcDNA3.1/Zeo(+)-B55β-HA, pcDNA3.1/Zeo(+)-B55βαβ-HA, pcDNA3.1/Zeo(+)-B55β2-HA, pcDNA3.1/Zeo(+)- B55δ-HA, or pcDNA3.1/Zeo(+)-B56γ3-HA. Expression of various exogenous B isoforms was assessed by indirect immunofluorescence using the anti-HA antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as follows: predominantly nuclear (N > C), homogenously distributed in both nucleus and cytoplasm (N∼C), and predominantly cytoplasmic (N

    Journal: PLoS ONE

    Article Title: Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

    doi: 10.1371/journal.pone.0116074

    Figure Lengend Snippet: Subcellular distribution of PP2A subunits. ( A ) NIH3T3 cells were transiently transfected with pCA2-6myc-PP2A/Aα or pCMV-HA-PP2Acα-YC, and expression of the exogenous Aα and Cα subunits was assessed by indirect immunofluorescence using anti-Myc tag and anti-HA antibodies, respectively, in conjunction with Cy3-conjugated secondary antibody. ( B ) Diagrams of B55α, B55β, and the B55βαβ chimera mutant are shown. ( C ) NIH3T3 cells were transiently transfected with pcDNA3.1/Zeo(+)-B55α-HA, pcDNA3.1/Zeo(+)-B55β-HA, pcDNA3.1/Zeo(+)-B55βαβ-HA, pcDNA3.1/Zeo(+)-B55β2-HA, pcDNA3.1/Zeo(+)- B55δ-HA, or pcDNA3.1/Zeo(+)-B56γ3-HA. Expression of various exogenous B isoforms was assessed by indirect immunofluorescence using the anti-HA antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars: 20 µm. Cells with different distribution patterns were scored as follows: predominantly nuclear (N > C), homogenously distributed in both nucleus and cytoplasm (N∼C), and predominantly cytoplasmic (N

    Article Snippet: Cells were then blocked with 5% BSA in PBS for 1 h, and subsequently incubated with anti-HA (Cell Signaling, 2367) or anti-Myc tag (Cell Signaling, 2278) antibody for 1 h, followed by incubation with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Transfection, Expressing, Immunofluorescence, Mutagenesis, Staining

    α4 facilitates formation of PP2A AC core enzyme. ( A ) Equal amounts of BiFC expression constructs encoding YN-Aα and PP2Acα-YC in the presence of equal amounts of pcDNA5/To-Flag-α4 WT , pcDNA5/To-Flag-α4 MUT , or empty vector were transfected into NIH3T3 cells. YFP signals due to BiFC of YN-Aα and PP2Acα-YC were measured by fluorescence microscopy. Expression of Flag-α4 WT or Flag-α4 MUT was verified using anti-FLAG antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars, 20 µm. ( B ) BiFC expression constructs encoding YN-Aα and HA-PP2Acα-YC and pcDNA5/To-Flag-α4 WT , pcDNA5/To-Flag-α4 MUT , or empty vector were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection, and immunoprecipitations were performed using anti-HA Sepharose. The cell lysates and anti-HA immunocomplexes were then analyzed by SDS-PAGE and Western blotting using the indicated antibodies.

    Journal: PLoS ONE

    Article Title: Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

    doi: 10.1371/journal.pone.0116074

    Figure Lengend Snippet: α4 facilitates formation of PP2A AC core enzyme. ( A ) Equal amounts of BiFC expression constructs encoding YN-Aα and PP2Acα-YC in the presence of equal amounts of pcDNA5/To-Flag-α4 WT , pcDNA5/To-Flag-α4 MUT , or empty vector were transfected into NIH3T3 cells. YFP signals due to BiFC of YN-Aα and PP2Acα-YC were measured by fluorescence microscopy. Expression of Flag-α4 WT or Flag-α4 MUT was verified using anti-FLAG antibody and Cy3-conjugated secondary antibody. DAPI was applied for staining of nuclei. Scale bars, 20 µm. ( B ) BiFC expression constructs encoding YN-Aα and HA-PP2Acα-YC and pcDNA5/To-Flag-α4 WT , pcDNA5/To-Flag-α4 MUT , or empty vector were transfected into NIH3T3 cells. Cell lysates were collected 24 h post-transfection, and immunoprecipitations were performed using anti-HA Sepharose. The cell lysates and anti-HA immunocomplexes were then analyzed by SDS-PAGE and Western blotting using the indicated antibodies.

    Article Snippet: Cells were then blocked with 5% BSA in PBS for 1 h, and subsequently incubated with anti-HA (Cell Signaling, 2367) or anti-Myc tag (Cell Signaling, 2278) antibody for 1 h, followed by incubation with Cy3-conjugated secondary antibodies (Jackson ImmunoResearch).

    Techniques: Bimolecular Fluorescence Complementation Assay, Expressing, Construct, Plasmid Preparation, Transfection, Fluorescence, Microscopy, Staining, SDS Page, Western Blot