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Olympus olympus fv1000
Distribution of fluorescence imaging. (A) S. typhimurium -A1-R-GFP targeting the osteosarcoma PDOX after intra-arterial (i.a.) injection. (B) S. typhimurium A1-R-GFP targeting the osteosarcoma after intravenous (i.v.) injection. Confocal microscopy imaging with the Olympus <t>FV1000</t> demonstrated S. typhimurium A1-R-GFP targeting the osteosarcoma PDOX. Scale bars: 12.5 µm.
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Images

1) Product Images from "Intra-arterial administration of tumor-targeting Salmonella typhimurium A1-R regresses a cisplatin-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model"

Article Title: Intra-arterial administration of tumor-targeting Salmonella typhimurium A1-R regresses a cisplatin-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model

Journal: Cell Cycle

doi: 10.1080/15384101.2017.1317417

Distribution of fluorescence imaging. (A) S. typhimurium -A1-R-GFP targeting the osteosarcoma PDOX after intra-arterial (i.a.) injection. (B) S. typhimurium A1-R-GFP targeting the osteosarcoma after intravenous (i.v.) injection. Confocal microscopy imaging with the Olympus FV1000 demonstrated S. typhimurium A1-R-GFP targeting the osteosarcoma PDOX. Scale bars: 12.5 µm.
Figure Legend Snippet: Distribution of fluorescence imaging. (A) S. typhimurium -A1-R-GFP targeting the osteosarcoma PDOX after intra-arterial (i.a.) injection. (B) S. typhimurium A1-R-GFP targeting the osteosarcoma after intravenous (i.v.) injection. Confocal microscopy imaging with the Olympus FV1000 demonstrated S. typhimurium A1-R-GFP targeting the osteosarcoma PDOX. Scale bars: 12.5 µm.

Techniques Used: Fluorescence, Imaging, Injection, Confocal Microscopy

2) Product Images from "Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria"

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria

Journal: PLoS ONE

doi: 10.1371/journal.pone.0002379

MAPKs and MAPKKs localize in tumor mitochondria. (A) LP07 cells were stained with MitoTracker Deep Red, fixed and immune stained with anti ERK1/2, JNK1/2 and p38 primary antibodies and secondary antibodies conjugated with Cy3, and analyzed in an Olympus FV1000 confocal microscope. Images directly exported from Olympus Fluoview acquisition program were processed with DIP image software for MATLAB, and a 2D fluorescence intensity histogram was performed. Pixel frequency map displayed on the right. Bar = 10 µm. (B) Submitochondrial localization of MAPKs and MAPKKs was assessed by western blot. (M: mitochondria; Mpl: mitoplast; OMM: outer mitochondrial membrane; IMS: intermembrane space; IMM: inner mitochondrial membrane; Mx: mitochondrial matrix). Identity of mitochondrial fractions was corroborated with specific antibodies anti complex I 39 kDa subunit, voltage-dependent anion channel (VDAC1), superoxide dismutase II and cytochrome oxidase, subunit VI C. (C) ERK1/2 was detected in LP07 mitochondria by immune labelling and transmission electron microscopy.
Figure Legend Snippet: MAPKs and MAPKKs localize in tumor mitochondria. (A) LP07 cells were stained with MitoTracker Deep Red, fixed and immune stained with anti ERK1/2, JNK1/2 and p38 primary antibodies and secondary antibodies conjugated with Cy3, and analyzed in an Olympus FV1000 confocal microscope. Images directly exported from Olympus Fluoview acquisition program were processed with DIP image software for MATLAB, and a 2D fluorescence intensity histogram was performed. Pixel frequency map displayed on the right. Bar = 10 µm. (B) Submitochondrial localization of MAPKs and MAPKKs was assessed by western blot. (M: mitochondria; Mpl: mitoplast; OMM: outer mitochondrial membrane; IMS: intermembrane space; IMM: inner mitochondrial membrane; Mx: mitochondrial matrix). Identity of mitochondrial fractions was corroborated with specific antibodies anti complex I 39 kDa subunit, voltage-dependent anion channel (VDAC1), superoxide dismutase II and cytochrome oxidase, subunit VI C. (C) ERK1/2 was detected in LP07 mitochondria by immune labelling and transmission electron microscopy.

Techniques Used: Staining, Microscopy, Software, Fluorescence, Western Blot, Transmission Assay, Electron Microscopy

3) Product Images from "Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo"

Article Title: Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0030775

Confocal images of transplanted cyto-GFP expressing cells in the s hiverer spinal cord. Four weeks after transplantation of cyto-GFP expressing neurospheres, fixed sections of the shiverer spinal cord were immunolabelled for MBP (red) and GFP (green). A ) A cyto-GFP labelled cell appears to extend spirals of cytoplasm around an MBP-positive myelin-like sheath (yellow arrows). Below the cell body, cyto-GFP is seen at the lateral edges (in relation to the long axis of the sheaths, white arrow) of adjacent sheaths and probably represents the cytoplasm filled paranodal loops on either side of the node of Ranvier (asterisks). B ) Schematic of visualisation of the sections in C and D. Ci–ii and Di–ii ) Spiral of GFP cytoplasm was followed by focussing up and down through the plane of view where they crossed up, traversed the axonal surface, then crossed down again representing the looping as shown in the schematic in B. E – G ) 3D reconstruction of cyto-GFP structures ( E ), illustrates cyto-GFP either side of a space typical of a node of Ranvier (white arrows). F ) is a tilted perspective of E) and shows the cyto-GFP form complete rings (white arrows representing the same position in E), consistent with the morphology of paranodal loops. I ) Asymmetric caspr positive structures in association with cyto-GFP, at either side of a heminode. On the left, caspr forms a single vertical line and co-localises with cyto-GFP from the myelinating cell. On the right, caspr appears like a loose coil, consistent with its pattern of expression in non-myelinated axons. All images were acquired using an Olympus FV1000 confocal microscope (×60, 1.35NA). Representative images from at least 10 separate experiments.
Figure Legend Snippet: Confocal images of transplanted cyto-GFP expressing cells in the s hiverer spinal cord. Four weeks after transplantation of cyto-GFP expressing neurospheres, fixed sections of the shiverer spinal cord were immunolabelled for MBP (red) and GFP (green). A ) A cyto-GFP labelled cell appears to extend spirals of cytoplasm around an MBP-positive myelin-like sheath (yellow arrows). Below the cell body, cyto-GFP is seen at the lateral edges (in relation to the long axis of the sheaths, white arrow) of adjacent sheaths and probably represents the cytoplasm filled paranodal loops on either side of the node of Ranvier (asterisks). B ) Schematic of visualisation of the sections in C and D. Ci–ii and Di–ii ) Spiral of GFP cytoplasm was followed by focussing up and down through the plane of view where they crossed up, traversed the axonal surface, then crossed down again representing the looping as shown in the schematic in B. E – G ) 3D reconstruction of cyto-GFP structures ( E ), illustrates cyto-GFP either side of a space typical of a node of Ranvier (white arrows). F ) is a tilted perspective of E) and shows the cyto-GFP form complete rings (white arrows representing the same position in E), consistent with the morphology of paranodal loops. I ) Asymmetric caspr positive structures in association with cyto-GFP, at either side of a heminode. On the left, caspr forms a single vertical line and co-localises with cyto-GFP from the myelinating cell. On the right, caspr appears like a loose coil, consistent with its pattern of expression in non-myelinated axons. All images were acquired using an Olympus FV1000 confocal microscope (×60, 1.35NA). Representative images from at least 10 separate experiments.

Techniques Used: Expressing, Transplantation Assay, Microscopy

Static images of myelination from mouse cultures in vitro using differentiation markers. A–B ) Confocal image acquired using FV1000 with 60× 0.75NA (A–C) showing the initial contact between oligodendrocytes (O4, red) and neurites (SMI-31, green) in a wild type mouse myelinating culture. After DIV 9 the oligodendrocyte's processes appear to align along neurites B ). Subsequent stages in axon/oligodendrocyte contact and wrapping where PLP/DM20 can be detected alongside expression of the O4 antibody. The staining suggests that myelin sheaths wrap around segments of the axon at this stage (solid regions of red and green; arrows) around DIV 9. C ) Image of a culture after 28 days in vitro . The oligodendrocyte's many processes appear to “fill-out” (solid green sheaths) from the initial spirals (arrows) of the membrane visualised by PLP/DM20 (green) around SMI-31 (red) neurites. D ) Single contiguous myelin sheaths begin to appear around 17–18 days in vitro using anti-PLP/DM20. Nodes of Ranvier are apparent between the internodes of myelin (asterisk) using an antibody to the nodal protein Caspr (insert). Image taken with and a Zeiss Axioplan II, 20×, 0.75NA (D) and similar images were observed from 5–10 separate experiments.
Figure Legend Snippet: Static images of myelination from mouse cultures in vitro using differentiation markers. A–B ) Confocal image acquired using FV1000 with 60× 0.75NA (A–C) showing the initial contact between oligodendrocytes (O4, red) and neurites (SMI-31, green) in a wild type mouse myelinating culture. After DIV 9 the oligodendrocyte's processes appear to align along neurites B ). Subsequent stages in axon/oligodendrocyte contact and wrapping where PLP/DM20 can be detected alongside expression of the O4 antibody. The staining suggests that myelin sheaths wrap around segments of the axon at this stage (solid regions of red and green; arrows) around DIV 9. C ) Image of a culture after 28 days in vitro . The oligodendrocyte's many processes appear to “fill-out” (solid green sheaths) from the initial spirals (arrows) of the membrane visualised by PLP/DM20 (green) around SMI-31 (red) neurites. D ) Single contiguous myelin sheaths begin to appear around 17–18 days in vitro using anti-PLP/DM20. Nodes of Ranvier are apparent between the internodes of myelin (asterisk) using an antibody to the nodal protein Caspr (insert). Image taken with and a Zeiss Axioplan II, 20×, 0.75NA (D) and similar images were observed from 5–10 separate experiments.

Techniques Used: In Vitro, Plasmid Purification, Expressing, Staining

4) Product Images from "Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy"

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy

Journal: Macromolecular bioscience

doi: 10.1002/mabi.201600333

Cellular uptake study by Confocal Microscopy Cells were grown on Lab-Tek™ Chambered Coverglass for 24 hours. After incubation for with drugs ((MTCP 0.158 mM, PAMAM-MTCP 20.61 µM, HPMA-MTCP 4.49 µM)) for 7hours, cell membranes were stained with Wheat germ agglutinin CF™488A conjugate and cell nuclei were stained with Hoechst according to the manufacturer’s protocol. Cells fixed with 4% para-formaldehyde solution in PBS. Red dots are represented to polymer-conjugated MTCP. Fluorescent images of fixed cells were taken under a confocal laser scanning microscope (Olympus FV1000 - US).
Figure Legend Snippet: Cellular uptake study by Confocal Microscopy Cells were grown on Lab-Tek™ Chambered Coverglass for 24 hours. After incubation for with drugs ((MTCP 0.158 mM, PAMAM-MTCP 20.61 µM, HPMA-MTCP 4.49 µM)) for 7hours, cell membranes were stained with Wheat germ agglutinin CF™488A conjugate and cell nuclei were stained with Hoechst according to the manufacturer’s protocol. Cells fixed with 4% para-formaldehyde solution in PBS. Red dots are represented to polymer-conjugated MTCP. Fluorescent images of fixed cells were taken under a confocal laser scanning microscope (Olympus FV1000 - US).

Techniques Used: Confocal Microscopy, Incubation, Staining, Laser-Scanning Microscopy

5) Product Images from "Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells"

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193022

Mitochondrial ERK localization depends on differential phosphorylation. Representative confocal images of LP07 tumor lung transfected cells with wild type ERK/V5 WT (left panel), Y183A/V5 (middle panel) and T183A/V5 (right panel). Forty-eight hours post transfection, cells were incubated with 1 μM H 2 O 2 during 0–30 min. From top to bottom of the panels images show increasing H 2 O 2 incubating times. Mitochondria were visualized in red by staining with Mitotracker Deep Red. Cells were fixed and incubated with anti-V5 antibody and a secondary antibody conjugated with Cy2, and analyzed in an Olympus FV1000 confocal microscope. Co-localization was highlighted in green on the grayscale images (last column of each panel). Images directly exported from Olympus Fluoview acquisition program. Fold changes relative to control images of combined MitoTracker and Cy2 intensity for ERK2 WT (Panel A), Y185A (Panel B) and T183A (Panel C). Basal conditions without H 2 O 2 arbitrarily defined as 1. Bar = 10 μm.
Figure Legend Snippet: Mitochondrial ERK localization depends on differential phosphorylation. Representative confocal images of LP07 tumor lung transfected cells with wild type ERK/V5 WT (left panel), Y183A/V5 (middle panel) and T183A/V5 (right panel). Forty-eight hours post transfection, cells were incubated with 1 μM H 2 O 2 during 0–30 min. From top to bottom of the panels images show increasing H 2 O 2 incubating times. Mitochondria were visualized in red by staining with Mitotracker Deep Red. Cells were fixed and incubated with anti-V5 antibody and a secondary antibody conjugated with Cy2, and analyzed in an Olympus FV1000 confocal microscope. Co-localization was highlighted in green on the grayscale images (last column of each panel). Images directly exported from Olympus Fluoview acquisition program. Fold changes relative to control images of combined MitoTracker and Cy2 intensity for ERK2 WT (Panel A), Y185A (Panel B) and T183A (Panel C). Basal conditions without H 2 O 2 arbitrarily defined as 1. Bar = 10 μm.

Techniques Used: Transfection, Incubation, Staining, Microscopy

6) Product Images from "Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways"

Article Title: Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddr203

IIS and HSF-1 pathways regulated mutant ataxin-3-mediated proteotoxicity in C. elegans neurons. ( A ) Flattened z-series of AT3q130 and daf-2 ; AT3q130 animals that were grown at 15°C for 4 days and transferred to 20°C for 24 h. ( B – E ) Animals were grown at 20°C during their lifespan. White squares highlight the area where the decrease in aggregation is most clear. (A, B and F ) daf-2(e1370) and age-1(hx546) mutations tend to reduce AT3q130-mediated aggregation. The absence of DAF-16 (D) and HSF-1 (E) increased aggregation of mutant ATXN3 (F). The daf-16(mu86) mutation caused a mild aggravation of the aggregation phenotype, visible at day 3 (post-hatching) (D, square). hsf-1(sy441) (E) mutation had a great impact on aggregation, with some aggregates visible already in embryos (arrow). Scale bar, 50 µm. All pictures were obtained using an Olympus FV1000 confocal microscope. (F) Quantification of the number of aggregates per area of animal of all strains. Data show the mean ± SD of eight or more animals. Asterisk indicates the significant mean difference between either hsf-1 ; AT3q130 or daf-16 ; AT3q130 and AT3q130 animals; hash symbol indicates the significant difference between hsf-1 ; AT3q130 and daf-16 ; AT3q130 ( ANOVA , applying Bonferroni correction with 95% confidence intervals; * ,# P
Figure Legend Snippet: IIS and HSF-1 pathways regulated mutant ataxin-3-mediated proteotoxicity in C. elegans neurons. ( A ) Flattened z-series of AT3q130 and daf-2 ; AT3q130 animals that were grown at 15°C for 4 days and transferred to 20°C for 24 h. ( B – E ) Animals were grown at 20°C during their lifespan. White squares highlight the area where the decrease in aggregation is most clear. (A, B and F ) daf-2(e1370) and age-1(hx546) mutations tend to reduce AT3q130-mediated aggregation. The absence of DAF-16 (D) and HSF-1 (E) increased aggregation of mutant ATXN3 (F). The daf-16(mu86) mutation caused a mild aggravation of the aggregation phenotype, visible at day 3 (post-hatching) (D, square). hsf-1(sy441) (E) mutation had a great impact on aggregation, with some aggregates visible already in embryos (arrow). Scale bar, 50 µm. All pictures were obtained using an Olympus FV1000 confocal microscope. (F) Quantification of the number of aggregates per area of animal of all strains. Data show the mean ± SD of eight or more animals. Asterisk indicates the significant mean difference between either hsf-1 ; AT3q130 or daf-16 ; AT3q130 and AT3q130 animals; hash symbol indicates the significant difference between hsf-1 ; AT3q130 and daf-16 ; AT3q130 ( ANOVA , applying Bonferroni correction with 95% confidence intervals; * ,# P

Techniques Used: Mutagenesis, Microscopy

7) Product Images from "Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells"

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193022

Mitochondrial ERK localization depends on differential phosphorylation. Representative confocal images of LP07 tumor lung transfected cells with wild type ERK/V5 WT (left panel), Y183A/V5 (middle panel) and T183A/V5 (right panel). Forty-eight hours post transfection, cells were incubated with 1 μM H 2 O 2 during 0–30 min. From top to bottom of the panels images show increasing H 2 O 2 incubating times. Mitochondria were visualized in red by staining with Mitotracker Deep Red. Cells were fixed and incubated with anti-V5 antibody and a secondary antibody conjugated with Cy2, and analyzed in an Olympus FV1000 confocal microscope. Co-localization was highlighted in green on the grayscale images (last column of each panel). Images directly exported from Olympus Fluoview acquisition program. Fold changes relative to control images of combined MitoTracker and Cy2 intensity for ERK2 WT (Panel A), Y185A (Panel B) and T183A (Panel C). Basal conditions without H 2 O 2 arbitrarily defined as 1. Bar = 10 μm.
Figure Legend Snippet: Mitochondrial ERK localization depends on differential phosphorylation. Representative confocal images of LP07 tumor lung transfected cells with wild type ERK/V5 WT (left panel), Y183A/V5 (middle panel) and T183A/V5 (right panel). Forty-eight hours post transfection, cells were incubated with 1 μM H 2 O 2 during 0–30 min. From top to bottom of the panels images show increasing H 2 O 2 incubating times. Mitochondria were visualized in red by staining with Mitotracker Deep Red. Cells were fixed and incubated with anti-V5 antibody and a secondary antibody conjugated with Cy2, and analyzed in an Olympus FV1000 confocal microscope. Co-localization was highlighted in green on the grayscale images (last column of each panel). Images directly exported from Olympus Fluoview acquisition program. Fold changes relative to control images of combined MitoTracker and Cy2 intensity for ERK2 WT (Panel A), Y185A (Panel B) and T183A (Panel C). Basal conditions without H 2 O 2 arbitrarily defined as 1. Bar = 10 μm.

Techniques Used: Transfection, Incubation, Staining, Microscopy

8) Product Images from "Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers"

Article Title: Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers

Journal: Transplantation

doi: 10.1097/TP.0000000000001071

Human platelet accumulation by GGTA1 −/− CMAH −/− , GGTA1 −/− , ASGR1 −/− , and WT liver sinusoidal endothelial cells in vitro Primary WT, GGTA1 −/− , ASGR1 −/− and CMAH −/− GGTA1 −/− LSEC lines were isolated and cultured for 4 days. Human platelets were isolated from a healthy donor and labeled with carboxyfluorescein succinimidyl ester (CFSE). Labeled Platelets (Green) were added to experimental wells containing primary endothelial cells. Cells were stained with anti-Pig CD31 (Red) to visualize the endothelial surface; and DAPI (Blue) was added to all slides as a nuclear stain. Confocal microscopy was performed using an Olympus FV1000. The relationships between human platelets and GGTA1 −/− CMAH −/− or GGTA1 −/− livers seen in the ex vivo perfusion model was upheld by in vitro analysis. Based on colocalization analysis, CFSE-labeled human platelets associated less with porcine liver endothelial cells from the GGTA1 −/− CMAH −/− background compared to WT (p=0.002) and GGTA1 −/− (p=0.002) cells; ASGR1 −/− LSECs exhibited significantly less platelet binding than GGTA1 −/− (p=0.013) and WT (p=0.009) cells. Although GGTA1 −/− CMAH −/− LSECs appeared to associate fewer human platelets than ASGR1 −/− LSECs, the difference failed to reach statistical significance (p= 0.8). Figure is representative of all images captured (n= 16 for each cohort).
Figure Legend Snippet: Human platelet accumulation by GGTA1 −/− CMAH −/− , GGTA1 −/− , ASGR1 −/− , and WT liver sinusoidal endothelial cells in vitro Primary WT, GGTA1 −/− , ASGR1 −/− and CMAH −/− GGTA1 −/− LSEC lines were isolated and cultured for 4 days. Human platelets were isolated from a healthy donor and labeled with carboxyfluorescein succinimidyl ester (CFSE). Labeled Platelets (Green) were added to experimental wells containing primary endothelial cells. Cells were stained with anti-Pig CD31 (Red) to visualize the endothelial surface; and DAPI (Blue) was added to all slides as a nuclear stain. Confocal microscopy was performed using an Olympus FV1000. The relationships between human platelets and GGTA1 −/− CMAH −/− or GGTA1 −/− livers seen in the ex vivo perfusion model was upheld by in vitro analysis. Based on colocalization analysis, CFSE-labeled human platelets associated less with porcine liver endothelial cells from the GGTA1 −/− CMAH −/− background compared to WT (p=0.002) and GGTA1 −/− (p=0.002) cells; ASGR1 −/− LSECs exhibited significantly less platelet binding than GGTA1 −/− (p=0.013) and WT (p=0.009) cells. Although GGTA1 −/− CMAH −/− LSECs appeared to associate fewer human platelets than ASGR1 −/− LSECs, the difference failed to reach statistical significance (p= 0.8). Figure is representative of all images captured (n= 16 for each cohort).

Techniques Used: In Vitro, Isolation, Cell Culture, Labeling, Staining, Confocal Microscopy, Ex Vivo, Binding Assay

Related Articles

Cytometry:

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: .. Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. For all methods statistical analyses were performed with Excel 2010 software (Microsoft, USA).

Incubation:

Article Title: Spontaneous and Acetylcholine Evoked Calcium Transients in the Developing Mouse Utricle
Article Snippet: Secondary antisera dilutions were added to tissues and incubated for 1 h at RT and subsequently rinsed in PBS as described for primary antisera. .. Images were obtained on Olympus FV1000 using a 60X oil immersion confocal microscope, images were post processed in FluoRender, and formatted for publication in Adobe Illustrator.

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: Fluorescence labeling and confocal microscopy Cells were grown on cover slides and stained with a specific mitochondrial marker, MitoTracker Deep Red 633 FM (Molecular probes) (100 nM, 45 min at 37°C), fixed in 4% paraformaldehyde, blocked in 1% BSA, 0.3% Triton X-100, PBS, pH 7.4, in a humidified chamber for 1 h, and incubated with primary (anti ERK1/2, JNK1/2 or p38) antibodies and secondary antibodies conjugated with Cy3 for 1h at RT in the same buffer. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Article Title: Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers
Article Snippet: Slides were incubated with secondary antibody for approximately 1 h and then washed three times with PBS. .. Confocal microscopy was performed using an Olympus FV1000 (Olympus America Inc., Center Valley, PA, USA).

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Forty-eight hours post-transfection, cells were incubated with H2 O2 as indicated in the figures, then stained with 100 nM specific mitochondrial marker MitoTracker Deep Red 633 FM (Molecular Probes, Thermo Fisher Scientific) for 45 min at 37°C, fixed in 4% paraformaldehyde, blocked in 1% BSA, 0.3% Triton X-100 PBS, pH 7.4, in a humidified chamber for 1 h, and incubated with primary antibody against V5 tag (anti-V5 tag) and secondary antibody conjugated with Cy3 (dilution 1:400; #111-165-003, Jackson ImmunoResearch Inc., West Grove, PA, USA) for 1h at room temperature in the same buffer. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective.

Expressing:

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: Determination of Photobleaching Rates Laser-scanning confocal and widefield microscopy photobleaching experiments utilized HeLa S3 cells (CCL2 line; ATCC) expressing fusions of pcFP fused with human histone H2B, as described above for the photoconversion experiments. .. For laser-scanning confocal photobleaching an Olympus FV1000 was used to first image the cells at a low magnification to ensure cell vitality.

Ex Vivo:

Article Title: Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers
Article Snippet: Duplicate wells for each group were washed with 150 μL of PBS three times then fixed with 50 μL of 4% paraformaldehyde–PBS solution for 20 min. To facilitate immunohistochemical visualization, and confirmation of platelet deposition within ex vivo platelet-perfused livers, punch biopsies were obtained at the conclusion of perfusions, sectioned at 4um and then fixed with 50 μL of 4% paraformaldehyde–PBS solution for 20 min. .. Confocal microscopy was performed using an Olympus FV1000 (Olympus America Inc., Center Valley, PA, USA).

Flow Cytometry:

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: .. Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. For all methods statistical analyses were performed with Excel 2010 software (Microsoft, USA).

Transfection:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: For in vivo imaging, cells were grown on Lab-Tek Chambered Borosilicate Coverglass System (Nunc), and transfected with GFP-hERK2 or JNK1-GFP, and stained with Mitotracker as described above. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Fluorescence labeling and confocal microscopy Cells were grown on cover slides and transfected as mentioned above. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective.

Immunolabeling:

Article Title: Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability
Article Snippet: Immunohistochemistry The brain and thoracicoabdominal ganglion were prepared for immunolabeling and analyzed as previously described , , . .. Frontal optical sections of brain samples were taken with confocal microscopy, Olympus FV1000 or Leica SP2.

Nuclear Magnetic Resonance:

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: .. Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. For all methods statistical analyses were performed with Excel 2010 software (Microsoft, USA).

Generated:

Article Title: Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways
Article Snippet: All images were captured either on a Zeiss LSM510 META (Oberkochen, Germany) or on an Olympus FV1000 (Japan) confocal microscope, under a 63× water or 60× oil objective, respectively. .. Z-series imaging was taken of all the C. elegans lines generated, using 514/515 nm laser excitation for YFP, 458 nm for CFP and 593 nm for mCherry fusion proteins.

Confocal Laser Scanning Microscopy:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective. ..

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
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Imaging:

Article Title: Identifying the In Vivo Cellular Correlates of Antipsychotic Drugs
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Article Snippet: Paragraph title: Confocal imaging ... All images were captured either on a Zeiss LSM510 META (Oberkochen, Germany) or on an Olympus FV1000 (Japan) confocal microscope, under a 63× water or 60× oil objective, respectively.

Article Title: Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo
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In Vivo Imaging:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: For in vivo imaging, cells were grown on Lab-Tek Chambered Borosilicate Coverglass System (Nunc), and transfected with GFP-hERK2 or JNK1-GFP, and stained with Mitotracker as described above. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Injection:

Article Title: Intra-arterial administration of tumor-targeting Salmonella typhimurium A1-R regresses a cisplatin-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model
Article Snippet: .. Distribution of S. typhimurium A1-R was imaged by confocal microscopy with the Olympus FV1000 on day 3 after injection of S. typhimurium A1-R ( ). .. The mean fluorescence intensity in the PDOX tumor on day 3 after intra-arterial (i.a.) injection of S. typhimurium A1-R was 1.76 × 106 compared with 1.49 × 105 after intravenous (i.v.) injection (p = 0.000013; ).

Fluorescence:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: Paragraph title: Fluorescence labeling and confocal microscopy ... Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Paragraph title: Fluorescence labeling and confocal microscopy ... Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective.

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: Nuclei with similar size and fluorescence intensity were chosen for photobleaching experiments. .. For laser-scanning confocal photobleaching an Olympus FV1000 was used to first image the cells at a low magnification to ensure cell vitality.

Article Title: Intra-arterial administration of tumor-targeting Salmonella typhimurium A1-R regresses a cisplatin-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model
Article Snippet: Distribution of S. typhimurium A1-R was imaged by confocal microscopy with the Olympus FV1000 on day 3 after injection of S. typhimurium A1-R ( ). .. The mean fluorescence intensity in the PDOX tumor on day 3 after intra-arterial (i.a.) injection of S. typhimurium A1-R was 1.76 × 106 compared with 1.49 × 105 after intravenous (i.v.) injection (p = 0.000013; ).

Immunohistochemistry:

Article Title: Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers
Article Snippet: Duplicate wells for each group were washed with 150 μL of PBS three times then fixed with 50 μL of 4% paraformaldehyde–PBS solution for 20 min. To facilitate immunohistochemical visualization, and confirmation of platelet deposition within ex vivo platelet-perfused livers, punch biopsies were obtained at the conclusion of perfusions, sectioned at 4um and then fixed with 50 μL of 4% paraformaldehyde–PBS solution for 20 min. .. Confocal microscopy was performed using an Olympus FV1000 (Olympus America Inc., Center Valley, PA, USA).

Article Title: Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability
Article Snippet: Paragraph title: Immunohistochemistry ... Frontal optical sections of brain samples were taken with confocal microscopy, Olympus FV1000 or Leica SP2.

Microscopy:

Article Title: Spontaneous and Acetylcholine Evoked Calcium Transients in the Developing Mouse Utricle
Article Snippet: .. Images were obtained on Olympus FV1000 using a 60X oil immersion confocal microscope, images were post processed in FluoRender, and formatted for publication in Adobe Illustrator. .. Live Tissue Imaging and ACh Stimulation Membranous labyrinths were micro-dissected from Gad2-G5-tdT temporal bones (P1-P15; and adults up to age P533), in cold glycerol-modified Ringer's (in mM: 26 NaHCO3 , 11 glucose, 250 glycerol, 2.5 KCl, 1.2 NaH2 PO4 , 1.2 MgCl2 and 2.4 CaCl2; pH 7.4) (Rabbitt et al., ).

Article Title: Identifying the In Vivo Cellular Correlates of Antipsychotic Drugs
Article Snippet: .. Image acquisition and analysis The brain slices were imaged on the Olympus FV1000, confocal microscope. ..

Article Title: Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways
Article Snippet: .. All images were captured either on a Zeiss LSM510 META (Oberkochen, Germany) or on an Olympus FV1000 (Japan) confocal microscope, under a 63× water or 60× oil objective, respectively. ..

Article Title: Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability
Article Snippet: Frontal optical sections of brain samples were taken with confocal microscopy, Olympus FV1000 or Leica SP2. .. For evaluating the effect of GAL80, brains to be compared were scanned with identical microscopy setting.

Article Title: mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
Article Snippet: Determination of Photobleaching Rates Laser-scanning confocal and widefield microscopy photobleaching experiments utilized HeLa S3 cells (CCL2 line; ATCC) expressing fusions of pcFP fused with human histone H2B, as described above for the photoconversion experiments. .. For laser-scanning confocal photobleaching an Olympus FV1000 was used to first image the cells at a low magnification to ensure cell vitality.

Article Title: Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo
Article Snippet: .. For detailed morphological analysis of axons, oligodendrocytes and myelin sheath formation, images were captured by laser scanning confocal microscopy using either an Olympus FV1000 or Zeiss 710 confocal microscope. .. Image processing and movies were made with FV10 ASW (Olympus, Essex, UK) Full Version Viewer software.

Size-exclusion Chromatography:

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: .. Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. For all methods statistical analyses were performed with Excel 2010 software (Microsoft, USA).

Marker:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: Fluorescence labeling and confocal microscopy Cells were grown on cover slides and stained with a specific mitochondrial marker, MitoTracker Deep Red 633 FM (Molecular probes) (100 nM, 45 min at 37°C), fixed in 4% paraformaldehyde, blocked in 1% BSA, 0.3% Triton X-100, PBS, pH 7.4, in a humidified chamber for 1 h, and incubated with primary (anti ERK1/2, JNK1/2 or p38) antibodies and secondary antibodies conjugated with Cy3 for 1h at RT in the same buffer. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Forty-eight hours post-transfection, cells were incubated with H2 O2 as indicated in the figures, then stained with 100 nM specific mitochondrial marker MitoTracker Deep Red 633 FM (Molecular Probes, Thermo Fisher Scientific) for 45 min at 37°C, fixed in 4% paraformaldehyde, blocked in 1% BSA, 0.3% Triton X-100 PBS, pH 7.4, in a humidified chamber for 1 h, and incubated with primary antibody against V5 tag (anti-V5 tag) and secondary antibody conjugated with Cy3 (dilution 1:400; #111-165-003, Jackson ImmunoResearch Inc., West Grove, PA, USA) for 1h at room temperature in the same buffer. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective.

Labeling:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: Paragraph title: Fluorescence labeling and confocal microscopy ... Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Paragraph title: Fluorescence labeling and confocal microscopy ... Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective.

Confocal Microscopy:

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: .. Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. For all methods statistical analyses were performed with Excel 2010 software (Microsoft, USA).

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: Paragraph title: Fluorescence labeling and confocal microscopy ... Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Article Title: Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers
Article Snippet: .. Confocal microscopy was performed using an Olympus FV1000 (Olympus America Inc., Center Valley, PA, USA). .. CFSE-labeled human platelets associate less with isolated porcine liver endothelial cells from the GGTA1−/− CMAH−/− background compared to GGTA1−/− and WT cells.

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Paragraph title: Fluorescence labeling and confocal microscopy ... Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective.

Article Title: Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability
Article Snippet: .. Frontal optical sections of brain samples were taken with confocal microscopy, Olympus FV1000 or Leica SP2. .. For evaluating the effect of GAL80, brains to be compared were scanned with identical microscopy setting.

Article Title: Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo
Article Snippet: .. For detailed morphological analysis of axons, oligodendrocytes and myelin sheath formation, images were captured by laser scanning confocal microscopy using either an Olympus FV1000 or Zeiss 710 confocal microscope. .. Image processing and movies were made with FV10 ASW (Olympus, Essex, UK) Full Version Viewer software.

Article Title: Intra-arterial administration of tumor-targeting Salmonella typhimurium A1-R regresses a cisplatin-resistant relapsed osteosarcoma in a patient-derived orthotopic xenograft (PDOX) mouse model
Article Snippet: .. Distribution of S. typhimurium A1-R was imaged by confocal microscopy with the Olympus FV1000 on day 3 after injection of S. typhimurium A1-R ( ). .. The mean fluorescence intensity in the PDOX tumor on day 3 after intra-arterial (i.a.) injection of S. typhimurium A1-R was 1.76 × 106 compared with 1.49 × 105 after intravenous (i.v.) injection (p = 0.000013; ).

Software:

Article Title: Identifying the In Vivo Cellular Correlates of Antipsychotic Drugs
Article Snippet: Image acquisition and analysis The brain slices were imaged on the Olympus FV1000, confocal microscope. .. Images were processed using ImageJ 1.47 (NIH) software and the number of cells in each image were counted using Cell profiler (Openware, Broad Institute Imaging Platform).

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: .. Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. For all methods statistical analyses were performed with Excel 2010 software (Microsoft, USA).

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective. .. Images were acquired with Olympus Fluoview FV10-ASW software and analyzed with DIP image software for MATLAB (TNO, Delft).

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective. .. Images were obtained with Olympus Fluoview FV10-ASW software and analyzed with DIPimage software (image processing toolbox for Matlab, Delft University of Technology, The Netherlands).

Article Title: Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability
Article Snippet: Frontal optical sections of brain samples were taken with confocal microscopy, Olympus FV1000 or Leica SP2. .. Images of the confocal stacks were analyzed with the open-source software Image-J .

Article Title: Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo
Article Snippet: For detailed morphological analysis of axons, oligodendrocytes and myelin sheath formation, images were captured by laser scanning confocal microscopy using either an Olympus FV1000 or Zeiss 710 confocal microscope. .. Image processing and movies were made with FV10 ASW (Olympus, Essex, UK) Full Version Viewer software.

Two Tailed Test:

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. Student's t test (two tailed, unpaired) was performed for statistical analysis.

Standard Deviation:

Article Title: Comparative Endocytosis Mechanisms and Anticancer Effect of HPMA copolymer- and PAMAM Dendrimer-MTCP Conjugates for Photodynamic Therapy
Article Snippet: Analysis for each instrument was performed with associated software products as listed below: GraphPad Prism 5 for cytotoxicity; Flowjo (Version 10) for flow cytometry; Olympus FV1000 for confocal microscopy; Astra™ 5.3.4.13 (Wyatt Technologies, Santa Barbara, CA) for SEC; VNMR J4.2A for NMR analysis; MassLynx 4.1 for ESI/MS and FlexControl 3.4 for MALDI-TOF analysis. .. All experiments were performed in triplicate, and the results were presented as mean/median ± standard deviation.

Staining:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: For in vivo imaging, cells were grown on Lab-Tek Chambered Borosilicate Coverglass System (Nunc), and transfected with GFP-hERK2 or JNK1-GFP, and stained with Mitotracker as described above. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

Article Title: Silencing porcine CMAH and GGTA1 genes significantly reduces xenogeneic consumption of human platelets by porcine livers
Article Snippet: Paragraph title: Staining and Confocal Microscopy ... Confocal microscopy was performed using an Olympus FV1000 (Olympus America Inc., Center Valley, PA, USA).

Article Title: Subcellular distribution of ERK phosphorylation in tyrosine and threonine depends on redox status in murine lung cells
Article Snippet: Forty-eight hours post-transfection, cells were incubated with H2 O2 as indicated in the figures, then stained with 100 nM specific mitochondrial marker MitoTracker Deep Red 633 FM (Molecular Probes, Thermo Fisher Scientific) for 45 min at 37°C, fixed in 4% paraformaldehyde, blocked in 1% BSA, 0.3% Triton X-100 PBS, pH 7.4, in a humidified chamber for 1 h, and incubated with primary antibody against V5 tag (anti-V5 tag) and secondary antibody conjugated with Cy3 (dilution 1:400; #111-165-003, Jackson ImmunoResearch Inc., West Grove, PA, USA) for 1h at room temperature in the same buffer. .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective.

Article Title: Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability
Article Snippet: For dopamine staining , brains were fixed with 0.6% glutaraldehyde in PBS for 30 min; the unreacted aldehyde groups were subsequently reduced with 0.1% (w/v) sodium tetraborhydrate. .. Frontal optical sections of brain samples were taken with confocal microscopy, Olympus FV1000 or Leica SP2.

Fluorsave:

Article Title: Tumor Cell Phenotype Is Sustained by Selective MAPK Oxidation in Mitochondria
Article Snippet: Cover slides were mounted in Fluorsave mounting media (Calbiochem). .. Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 63×1.35 NA oil immersion objective.

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  • 99
    Olympus fluoview fv1000 confocal laser scanning microscope
    Confocal microscope image of TLR4, MD-2, TLR2, TLR5 and COX-2 expression in HT-29 cells with and without C. concisu infection. HT-29 cells were incubated with a representative C. concisus strain (P1CDB1) for 24 hours. Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells with and without C. concisus infection were detected by immunostained using specific antibodies, followed by Alex Fluor conjugated secondary antibodies. The image was viewed using an Olympus <t>FluoView</t> <t>FV1000</t> Confocal laser scanning microscope. The secondary antibodies used for detection of TLR4 and MD-2 were conjugated with Alexa Fluor 594 (emission colour red). The secondary antibodies used for detection of TLR2, TLR5 and COX-2 were conjugated with Alexa Fluor 488 (emission colour green). Scale Bar = 10 µm.
    Fluoview Fv1000 Confocal Laser Scanning Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 99/100, based on 354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluoview fv1000 confocal laser scanning microscope/product/Olympus
    Average 99 stars, based on 354 article reviews
    Price from $9.99 to $1999.99
    fluoview fv1000 confocal laser scanning microscope - by Bioz Stars, 2020-04
    99/100 stars
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    95
    Olympus confocal microscope olympus fluoview fv1000
    The fluorescent emission spectra are not affected by the protein fusion. mRFP fused to CDKA;1 ( A ) and free mRFP ( B ) were transiently expressed in tobacco and the fluorescence emission spectra were measured in the nucleus (marked by a ring) with Olympus <t>FluoView™</t> <t>FV1000</t> and Zeiss LSM 710 ( C ). Scale bars, 30 μm.
    Confocal Microscope Olympus Fluoview Fv1000, supplied by Olympus, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/confocal microscope olympus fluoview fv1000/product/Olympus
    Average 95 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    confocal microscope olympus fluoview fv1000 - by Bioz Stars, 2020-04
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    94
    Olympus laser confocal scanning olympus fv1000d upright microscope bx61
    9D5 antibody specific for dsRNA. A549 cells were infected with JUNV at an MOI of 1.0, At 48 hpi, infected and uninfected cell monolayers were untreated (A,D) or treated with RNase I (B) and RNAse III (C) . (A–D) Cells were then stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, red) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus <t>FV1000D</t> Upright Microscope <t>BX61</t> using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was done using the FIJI software. Laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Images shown are representative from three separate experiments.
    Laser Confocal Scanning Olympus Fv1000d Upright Microscope Bx61, supplied by Olympus, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/laser confocal scanning olympus fv1000d upright microscope bx61/product/Olympus
    Average 94 stars, based on 2 article reviews
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    laser confocal scanning olympus fv1000d upright microscope bx61 - by Bioz Stars, 2020-04
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    99
    Olympus olympus fv1000 microscope
    Accumulation of Hh on ILVs inside MVBs is necessary for its exovesicular secretion. (A) HhGFP-transfected S2R+ cells labeled with an endocytic pulse (2 h) of A647–anti-GFP Fab (left; red in merge) to localize endocytosed Hh and immunostained with anti-Hrs (middle; green in merge). A single confocal slice (for a 3D projection, see Supplemental Video S1). Note that A647–anti-GFP Fab staining is detected in enlarged endosomal organelles decorated with Hrs (arrowheads). Scale bar, 2 μm. (B, C) S2R+ cells were cotransfected with HhmCFP and LAMP-GFP (B) or with HhmCFP alone (C) and 2 d later incubated with A647–anti-GFP Fab singly for 20 min (B) or along with TMR-Dex for 2 h to localize organelles accessed by endocytic probes and imaged using a PerkinElmer spinning-disk confocal microscope (B) or an Olympus <t>FV1000</t> confocal microscope (C). Images represent a single confocal slice (for a 3D projection of the cells, see Supplemental Video S2). Note that endocytosed Hh (left; red in merge) colocalizes with vesicular LAMP-GFP (B; middle; green in merge) and with TMR-Dex (C; middle; green in merge) only in vesicular compartments but is segregated from tubular structures representing lysosomes marked by tubular TMR-Dex. Scale bar, 2 μm. (D) Unlike Hh-mCFP, endocytosed GFP-GPI (single confocal slice) visualized with a 2-h endocytic pulse of A647–anti-GFP Fab and imaged using an FV1000 confocal microscope is visualized in both tubular and vesicular structures of GFP-GPI–expressing S2R+ cells. Scale bar, 2 μm. (E) Immuno-EM on frozen sections from S2R+ cells transfected with HhGFP show labeling on the ILVs inside the MVB lumen, detected using the anti-Hh antibody and probed using 10-nm protein A–gold. (ii) Zoomed image of the MVB marked in (i); (iii, iv) other representative images of MVBs from different sections. Scale bar, 200 nm. (F, G) Western blots (F) show the amount of HhGFP in the corresponding cellular (Cells) and secreted fractions (P100, P250) obtained from HhGFP-transfected S2R+ cells treated with control (Zeocin) or with indicated RNAi after being subjected to the same sedimentation scheme shown in Figure 1A . Black vertical lines denote deletion of other lanes from the blots containing biological replicates of the same experiment. (G) Bar graph shows the extent of reduction in the amount of secreted HhGFP in P100 and P250 fractions (represented as percentage reduction in normalized secretion with respect to control; red dashed line) in the indicated RNAi treatment determined after quantifying the density of the staining in the Western blots. Nonsaturating exposures of cellular, P100, and P250 fractions were used for intensity measurements, and values in P100 and P250 fractions were normalized to the values in cell lysates. Data from at least three independent experiments are expressed as mean ± SD; * p
    Olympus Fv1000 Microscope, supplied by Olympus, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Confocal microscope image of TLR4, MD-2, TLR2, TLR5 and COX-2 expression in HT-29 cells with and without C. concisu infection. HT-29 cells were incubated with a representative C. concisus strain (P1CDB1) for 24 hours. Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells with and without C. concisus infection were detected by immunostained using specific antibodies, followed by Alex Fluor conjugated secondary antibodies. The image was viewed using an Olympus FluoView FV1000 Confocal laser scanning microscope. The secondary antibodies used for detection of TLR4 and MD-2 were conjugated with Alexa Fluor 594 (emission colour red). The secondary antibodies used for detection of TLR2, TLR5 and COX-2 were conjugated with Alexa Fluor 488 (emission colour green). Scale Bar = 10 µm.

    Journal: PLoS ONE

    Article Title: The Effects of Oral and Enteric Campylobacter concisus Strains on Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 Cells

    doi: 10.1371/journal.pone.0056888

    Figure Lengend Snippet: Confocal microscope image of TLR4, MD-2, TLR2, TLR5 and COX-2 expression in HT-29 cells with and without C. concisu infection. HT-29 cells were incubated with a representative C. concisus strain (P1CDB1) for 24 hours. Expression of TLR4, MD-2, TLR2, TLR5 and COX-2 in HT-29 cells with and without C. concisus infection were detected by immunostained using specific antibodies, followed by Alex Fluor conjugated secondary antibodies. The image was viewed using an Olympus FluoView FV1000 Confocal laser scanning microscope. The secondary antibodies used for detection of TLR4 and MD-2 were conjugated with Alexa Fluor 594 (emission colour red). The secondary antibodies used for detection of TLR2, TLR5 and COX-2 were conjugated with Alexa Fluor 488 (emission colour green). Scale Bar = 10 µm.

    Article Snippet: The cover slips were mounted using AF1 antifadent (Citifluor Ltd, London, UK) and HT-29 cells were observed using an Olympus FluoView FV1000 Confocal laser scanning microscope.

    Techniques: Microscopy, Expressing, Infection, Incubation, Laser-Scanning Microscopy

    The fluorescent emission spectra are not affected by the protein fusion. mRFP fused to CDKA;1 ( A ) and free mRFP ( B ) were transiently expressed in tobacco and the fluorescence emission spectra were measured in the nucleus (marked by a ring) with Olympus FluoView™ FV1000 and Zeiss LSM 710 ( C ). Scale bars, 30 μm.

    Journal: Plant Methods

    Article Title: Emission spectra profiling of fluorescent proteins in living plant cells

    doi: 10.1186/1746-4811-9-10

    Figure Lengend Snippet: The fluorescent emission spectra are not affected by the protein fusion. mRFP fused to CDKA;1 ( A ) and free mRFP ( B ) were transiently expressed in tobacco and the fluorescence emission spectra were measured in the nucleus (marked by a ring) with Olympus FluoView™ FV1000 and Zeiss LSM 710 ( C ). Scale bars, 30 μm.

    Article Snippet: Confocal microscopy, emission spectra analysis and linear unmixing Lambda stacks for each fluorophore were acquired with a confocal microscope Olympus FluoView™ FV1000 (Tokyo, Japan), with a 63× water corrected objective (numerical aperture of 1.2).

    Techniques: Fluorescence

    9D5 antibody specific for dsRNA. A549 cells were infected with JUNV at an MOI of 1.0, At 48 hpi, infected and uninfected cell monolayers were untreated (A,D) or treated with RNase I (B) and RNAse III (C) . (A–D) Cells were then stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, red) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was done using the FIJI software. Laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Images shown are representative from three separate experiments.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Visualization of Double-Stranded RNA Colocalizing With Pattern Recognition Receptors in Arenavirus Infected Cells

    doi: 10.3389/fcimb.2018.00251

    Figure Lengend Snippet: 9D5 antibody specific for dsRNA. A549 cells were infected with JUNV at an MOI of 1.0, At 48 hpi, infected and uninfected cell monolayers were untreated (A,D) or treated with RNase I (B) and RNAse III (C) . (A–D) Cells were then stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, red) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was done using the FIJI software. Laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Images shown are representative from three separate experiments.

    Article Snippet: Coverslips were imaged on a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens.

    Techniques: Infection, Staining, Microscopy, Software

    Detection of dsRNA in JUNV-infected A549 cells . (A) A549 cells were infected with the vaccine rCandid strain of JUNV at a MOI of 1.0. At 48 hpi, infected (JUNV) and uninfected (Mock) cell monolayers were fixed, permeabilized, and stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, red) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was done using the FIJI software. Laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Data shown are representative images from three separate experiments. Fluorescence plot profile of NP and dsRNA signal intensities around the white line is shown in (B) . (C) Quantitative analysis of the colocalization between dsRNA and NP signals using the Pearson's correlation coefficient in 100 infected cells from three separate experiments. The average and Std are given.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Visualization of Double-Stranded RNA Colocalizing With Pattern Recognition Receptors in Arenavirus Infected Cells

    doi: 10.3389/fcimb.2018.00251

    Figure Lengend Snippet: Detection of dsRNA in JUNV-infected A549 cells . (A) A549 cells were infected with the vaccine rCandid strain of JUNV at a MOI of 1.0. At 48 hpi, infected (JUNV) and uninfected (Mock) cell monolayers were fixed, permeabilized, and stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, red) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was done using the FIJI software. Laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Data shown are representative images from three separate experiments. Fluorescence plot profile of NP and dsRNA signal intensities around the white line is shown in (B) . (C) Quantitative analysis of the colocalization between dsRNA and NP signals using the Pearson's correlation coefficient in 100 infected cells from three separate experiments. The average and Std are given.

    Article Snippet: Coverslips were imaged on a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens.

    Techniques: Infection, Staining, Microscopy, Software, Fluorescence

    PKR recognizes JUNV dsRNA resulting in activation . (A,C) A549 cells were infected with rCandid at a MOI of 1.0. At 48 hpi, infected and uninfected cell monolayers were fixed, permeabilized, and stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, magenta), anti-PKR (Y117, red) (A) or anti-p-PKR (Thr-451, red) (C) and DAPI (blue). Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was done using the FIJI software. Laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. (B) Fluorescence plot profile of PKR, NP and dsRNA signal intensities around the white line is shown in the inlet. (D,E) Quantification of the colocalization between dsRNA (D) or NP (E) and p-PKR using the Pearson's correlation coefficient. 100 infected cells from three separate experiments were randomly selected and analyzed. Data shown is the average and Std.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Visualization of Double-Stranded RNA Colocalizing With Pattern Recognition Receptors in Arenavirus Infected Cells

    doi: 10.3389/fcimb.2018.00251

    Figure Lengend Snippet: PKR recognizes JUNV dsRNA resulting in activation . (A,C) A549 cells were infected with rCandid at a MOI of 1.0. At 48 hpi, infected and uninfected cell monolayers were fixed, permeabilized, and stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, magenta), anti-PKR (Y117, red) (A) or anti-p-PKR (Thr-451, red) (C) and DAPI (blue). Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was done using the FIJI software. Laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. (B) Fluorescence plot profile of PKR, NP and dsRNA signal intensities around the white line is shown in the inlet. (D,E) Quantification of the colocalization between dsRNA (D) or NP (E) and p-PKR using the Pearson's correlation coefficient. 100 infected cells from three separate experiments were randomly selected and analyzed. Data shown is the average and Std.

    Article Snippet: Coverslips were imaged on a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens.

    Techniques: Activation Assay, Infection, Staining, Microscopy, Software, Fluorescence

    RLRs, RIG-I and MDA-5 recognize JUNV dsRNA. (A,B,E) A549 cells were infected with rCandid at a MOI of 1.0. 48 hpi infected and uninfected cell monolayers were fixed, permeabilized, and stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, magenta), anti-RIG-I (red) (A,B) or anti-MDA-5 (red) (E) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was performed using the FIJI software. Unless indicated, laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Representative images of three separate experiment are shown. (B) Exposure of RIG-I in mock infected cells was increased to show the distribution pattern of RIG-I in uninfected cells. (C,F) Quantification of the colocalization between dsRNA and RIG-I (C) or MDA-5 (F) using the Pearson's correlation coefficient. (D,G) Quantification of the colocalization between NP and RIG-I (D) or MDA-5 (G) . Data shown represents the average and Std of 100 infected cells from three separate experiments.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Visualization of Double-Stranded RNA Colocalizing With Pattern Recognition Receptors in Arenavirus Infected Cells

    doi: 10.3389/fcimb.2018.00251

    Figure Lengend Snippet: RLRs, RIG-I and MDA-5 recognize JUNV dsRNA. (A,B,E) A549 cells were infected with rCandid at a MOI of 1.0. 48 hpi infected and uninfected cell monolayers were fixed, permeabilized, and stained with the anti-dsRNA antibody (9D5, green), anti-JUNV NP (AG12, magenta), anti-RIG-I (red) (A,B) or anti-MDA-5 (red) (E) and DAPI (blue) as described in the Materials and Methods section. Cells were then observed under a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens. All image analysis and processing was performed using the FIJI software. Unless indicated, laser emissions were the same for all samples and only the same linear adjustments for brightness and contrast were made when appropriate across all samples. Representative images of three separate experiment are shown. (B) Exposure of RIG-I in mock infected cells was increased to show the distribution pattern of RIG-I in uninfected cells. (C,F) Quantification of the colocalization between dsRNA and RIG-I (C) or MDA-5 (F) using the Pearson's correlation coefficient. (D,G) Quantification of the colocalization between NP and RIG-I (D) or MDA-5 (G) . Data shown represents the average and Std of 100 infected cells from three separate experiments.

    Article Snippet: Coverslips were imaged on a Laser Confocal Scanning Olympus FV1000D Upright Microscope BX61 using 60x/1.42 numerical aperture oil immersion lens.

    Techniques: Multiple Displacement Amplification, Infection, Staining, Microscopy, Software

    Accumulation of Hh on ILVs inside MVBs is necessary for its exovesicular secretion. (A) HhGFP-transfected S2R+ cells labeled with an endocytic pulse (2 h) of A647–anti-GFP Fab (left; red in merge) to localize endocytosed Hh and immunostained with anti-Hrs (middle; green in merge). A single confocal slice (for a 3D projection, see Supplemental Video S1). Note that A647–anti-GFP Fab staining is detected in enlarged endosomal organelles decorated with Hrs (arrowheads). Scale bar, 2 μm. (B, C) S2R+ cells were cotransfected with HhmCFP and LAMP-GFP (B) or with HhmCFP alone (C) and 2 d later incubated with A647–anti-GFP Fab singly for 20 min (B) or along with TMR-Dex for 2 h to localize organelles accessed by endocytic probes and imaged using a PerkinElmer spinning-disk confocal microscope (B) or an Olympus FV1000 confocal microscope (C). Images represent a single confocal slice (for a 3D projection of the cells, see Supplemental Video S2). Note that endocytosed Hh (left; red in merge) colocalizes with vesicular LAMP-GFP (B; middle; green in merge) and with TMR-Dex (C; middle; green in merge) only in vesicular compartments but is segregated from tubular structures representing lysosomes marked by tubular TMR-Dex. Scale bar, 2 μm. (D) Unlike Hh-mCFP, endocytosed GFP-GPI (single confocal slice) visualized with a 2-h endocytic pulse of A647–anti-GFP Fab and imaged using an FV1000 confocal microscope is visualized in both tubular and vesicular structures of GFP-GPI–expressing S2R+ cells. Scale bar, 2 μm. (E) Immuno-EM on frozen sections from S2R+ cells transfected with HhGFP show labeling on the ILVs inside the MVB lumen, detected using the anti-Hh antibody and probed using 10-nm protein A–gold. (ii) Zoomed image of the MVB marked in (i); (iii, iv) other representative images of MVBs from different sections. Scale bar, 200 nm. (F, G) Western blots (F) show the amount of HhGFP in the corresponding cellular (Cells) and secreted fractions (P100, P250) obtained from HhGFP-transfected S2R+ cells treated with control (Zeocin) or with indicated RNAi after being subjected to the same sedimentation scheme shown in Figure 1A . Black vertical lines denote deletion of other lanes from the blots containing biological replicates of the same experiment. (G) Bar graph shows the extent of reduction in the amount of secreted HhGFP in P100 and P250 fractions (represented as percentage reduction in normalized secretion with respect to control; red dashed line) in the indicated RNAi treatment determined after quantifying the density of the staining in the Western blots. Nonsaturating exposures of cellular, P100, and P250 fractions were used for intensity measurements, and values in P100 and P250 fractions were normalized to the values in cell lysates. Data from at least three independent experiments are expressed as mean ± SD; * p

    Journal: Molecular Biology of the Cell

    Article Title: Oligomerization and endocytosis of Hedgehog is necessary for its efficient exovesicular secretion

    doi: 10.1091/mbc.E15-09-0671

    Figure Lengend Snippet: Accumulation of Hh on ILVs inside MVBs is necessary for its exovesicular secretion. (A) HhGFP-transfected S2R+ cells labeled with an endocytic pulse (2 h) of A647–anti-GFP Fab (left; red in merge) to localize endocytosed Hh and immunostained with anti-Hrs (middle; green in merge). A single confocal slice (for a 3D projection, see Supplemental Video S1). Note that A647–anti-GFP Fab staining is detected in enlarged endosomal organelles decorated with Hrs (arrowheads). Scale bar, 2 μm. (B, C) S2R+ cells were cotransfected with HhmCFP and LAMP-GFP (B) or with HhmCFP alone (C) and 2 d later incubated with A647–anti-GFP Fab singly for 20 min (B) or along with TMR-Dex for 2 h to localize organelles accessed by endocytic probes and imaged using a PerkinElmer spinning-disk confocal microscope (B) or an Olympus FV1000 confocal microscope (C). Images represent a single confocal slice (for a 3D projection of the cells, see Supplemental Video S2). Note that endocytosed Hh (left; red in merge) colocalizes with vesicular LAMP-GFP (B; middle; green in merge) and with TMR-Dex (C; middle; green in merge) only in vesicular compartments but is segregated from tubular structures representing lysosomes marked by tubular TMR-Dex. Scale bar, 2 μm. (D) Unlike Hh-mCFP, endocytosed GFP-GPI (single confocal slice) visualized with a 2-h endocytic pulse of A647–anti-GFP Fab and imaged using an FV1000 confocal microscope is visualized in both tubular and vesicular structures of GFP-GPI–expressing S2R+ cells. Scale bar, 2 μm. (E) Immuno-EM on frozen sections from S2R+ cells transfected with HhGFP show labeling on the ILVs inside the MVB lumen, detected using the anti-Hh antibody and probed using 10-nm protein A–gold. (ii) Zoomed image of the MVB marked in (i); (iii, iv) other representative images of MVBs from different sections. Scale bar, 200 nm. (F, G) Western blots (F) show the amount of HhGFP in the corresponding cellular (Cells) and secreted fractions (P100, P250) obtained from HhGFP-transfected S2R+ cells treated with control (Zeocin) or with indicated RNAi after being subjected to the same sedimentation scheme shown in Figure 1A . Black vertical lines denote deletion of other lanes from the blots containing biological replicates of the same experiment. (G) Bar graph shows the extent of reduction in the amount of secreted HhGFP in P100 and P250 fractions (represented as percentage reduction in normalized secretion with respect to control; red dashed line) in the indicated RNAi treatment determined after quantifying the density of the staining in the Western blots. Nonsaturating exposures of cellular, P100, and P250 fractions were used for intensity measurements, and values in P100 and P250 fractions were normalized to the values in cell lysates. Data from at least three independent experiments are expressed as mean ± SD; * p

    Article Snippet: Discs were stained using the primary antibodies overnight followed by washes using PBTx secondary antibodies for 2 h. Discs were washed after secondary antibody staining in PBTx and mounted in Vector shield mounting medium (Vector Laboratories, Burlingame, CA) and imaged using the Olympus FV1000 microscope.

    Techniques: Transfection, Labeling, Staining, Incubation, Microscopy, Expressing, Western Blot, Sedimentation

    Oligomerization-defective Hh is impaired in its endocytic delivery to the MVBs. (A) Model to explain the differences in trafficking of Hh variants to account for observed differences in secretion. Hh is represented in green as a homo-oligomer, and K132D is shown in blue in a monomeric form. The lengths of the arrows reflect the extent of traffic for each variant in a particular pathway. The extent of partitioning toward the recycling pathway of the Hh variants vs. the late endosomal pathway regulate delivery to the MVB and hence result in modulation of ILV formation. Alternatively, a more upstream endocytic block could also lead to similar consequences. (B, C) S2R+ cells expressing HhK132DmCFP were incubated with A647–anti-GFP Fab (red in merge) for 20 min (B) or 2 h (C) to localize internalized protein. It colocalizes with LAMP-GFP (B; green in merge) coexpressed in the same cells or with immunodetected Hrs (C; green in merge). Single confocal slices are shown from cells imaged using a PerkinElmer spinning-disk confocal microscope (B) or an Olympus FV1000 confocal microscope (C). Note that HhK132DmCFP traffics to LE/MVB structures and results in a redistribution of Hrs. Scale bar, 2 μm. (D) HhK132DmCFP is also visualized on ILVs inside the MVB lumen in cryosections of cells transfected with HhK132DmCFP labeled using anti-GFP antibody and detected using a 10-nm gold–conjugated secondary antibody. Scale bar, 200 nm. (E) Bar graphs showing the fraction of the surface-bound A647–anti-GFP Fab internalized in a 10-min endocytic pulse using a surface accessibility assay for each of the indicated Hh variants. Data from two experiments are represented as mean ± SEM from at least 50 cells for each variant; ** p

    Journal: Molecular Biology of the Cell

    Article Title: Oligomerization and endocytosis of Hedgehog is necessary for its efficient exovesicular secretion

    doi: 10.1091/mbc.E15-09-0671

    Figure Lengend Snippet: Oligomerization-defective Hh is impaired in its endocytic delivery to the MVBs. (A) Model to explain the differences in trafficking of Hh variants to account for observed differences in secretion. Hh is represented in green as a homo-oligomer, and K132D is shown in blue in a monomeric form. The lengths of the arrows reflect the extent of traffic for each variant in a particular pathway. The extent of partitioning toward the recycling pathway of the Hh variants vs. the late endosomal pathway regulate delivery to the MVB and hence result in modulation of ILV formation. Alternatively, a more upstream endocytic block could also lead to similar consequences. (B, C) S2R+ cells expressing HhK132DmCFP were incubated with A647–anti-GFP Fab (red in merge) for 20 min (B) or 2 h (C) to localize internalized protein. It colocalizes with LAMP-GFP (B; green in merge) coexpressed in the same cells or with immunodetected Hrs (C; green in merge). Single confocal slices are shown from cells imaged using a PerkinElmer spinning-disk confocal microscope (B) or an Olympus FV1000 confocal microscope (C). Note that HhK132DmCFP traffics to LE/MVB structures and results in a redistribution of Hrs. Scale bar, 2 μm. (D) HhK132DmCFP is also visualized on ILVs inside the MVB lumen in cryosections of cells transfected with HhK132DmCFP labeled using anti-GFP antibody and detected using a 10-nm gold–conjugated secondary antibody. Scale bar, 200 nm. (E) Bar graphs showing the fraction of the surface-bound A647–anti-GFP Fab internalized in a 10-min endocytic pulse using a surface accessibility assay for each of the indicated Hh variants. Data from two experiments are represented as mean ± SEM from at least 50 cells for each variant; ** p

    Article Snippet: Discs were stained using the primary antibodies overnight followed by washes using PBTx secondary antibodies for 2 h. Discs were washed after secondary antibody staining in PBTx and mounted in Vector shield mounting medium (Vector Laboratories, Burlingame, CA) and imaged using the Olympus FV1000 microscope.

    Techniques: Variant Assay, Blocking Assay, Expressing, Incubation, Microscopy, Transfection, Labeling