hoechst 33258  (Millipore)


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    Structured Review

    Millipore hoechst 33258
    Neutrophil transmigration in murine lungs induced by E. coli OMVs. Wild-type mice were intraperitoneally administered with either PBS or E. coli OMVs (15 μg in total protein amounts per mouse). Five animals were used in each group. Different groups of mice administered with either PBS or OMVs were killed at 6 h after OMV administration, and the lung tissues as well as BAL fluids were obtained from the five mice. (A–C) The lung sections of five mice were immunostained with anti-NIMP-R14 (green; neutrophils) and anti-CD31 (red; endothelial cells) antibodies (A) , or anti-NIMP-R14 (green; neutrophils) and anti-SP-C (red; lung epithelial cells) antibodies (B) . The sections were then counterstained with <t>Hoechst</t> 33258 (blue; nuclei). Representative fluorescence images are shown here. Scale bars = 50 μm. The number of neutrophils per field was counted from five confocal microscopy images obtained from the lung sections of five mice (C) . (D) The concentration of CXCL1 was measured in the BAL fluid by ELISA ( n = 5). Data were represented as mean ± SEM. ∗∗∗ P
    Hoechst 33258, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 928 article reviews
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    hoechst 33258 - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Outer Membrane Vesicles Derived From Escherichia coli Regulate Neutrophil Migration by Induction of Endothelial IL-8"

    Article Title: Outer Membrane Vesicles Derived From Escherichia coli Regulate Neutrophil Migration by Induction of Endothelial IL-8

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02268

    Neutrophil transmigration in murine lungs induced by E. coli OMVs. Wild-type mice were intraperitoneally administered with either PBS or E. coli OMVs (15 μg in total protein amounts per mouse). Five animals were used in each group. Different groups of mice administered with either PBS or OMVs were killed at 6 h after OMV administration, and the lung tissues as well as BAL fluids were obtained from the five mice. (A–C) The lung sections of five mice were immunostained with anti-NIMP-R14 (green; neutrophils) and anti-CD31 (red; endothelial cells) antibodies (A) , or anti-NIMP-R14 (green; neutrophils) and anti-SP-C (red; lung epithelial cells) antibodies (B) . The sections were then counterstained with Hoechst 33258 (blue; nuclei). Representative fluorescence images are shown here. Scale bars = 50 μm. The number of neutrophils per field was counted from five confocal microscopy images obtained from the lung sections of five mice (C) . (D) The concentration of CXCL1 was measured in the BAL fluid by ELISA ( n = 5). Data were represented as mean ± SEM. ∗∗∗ P
    Figure Legend Snippet: Neutrophil transmigration in murine lungs induced by E. coli OMVs. Wild-type mice were intraperitoneally administered with either PBS or E. coli OMVs (15 μg in total protein amounts per mouse). Five animals were used in each group. Different groups of mice administered with either PBS or OMVs were killed at 6 h after OMV administration, and the lung tissues as well as BAL fluids were obtained from the five mice. (A–C) The lung sections of five mice were immunostained with anti-NIMP-R14 (green; neutrophils) and anti-CD31 (red; endothelial cells) antibodies (A) , or anti-NIMP-R14 (green; neutrophils) and anti-SP-C (red; lung epithelial cells) antibodies (B) . The sections were then counterstained with Hoechst 33258 (blue; nuclei). Representative fluorescence images are shown here. Scale bars = 50 μm. The number of neutrophils per field was counted from five confocal microscopy images obtained from the lung sections of five mice (C) . (D) The concentration of CXCL1 was measured in the BAL fluid by ELISA ( n = 5). Data were represented as mean ± SEM. ∗∗∗ P

    Techniques Used: Transmigration Assay, Mouse Assay, Fluorescence, Confocal Microscopy, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Suppression of OMV-induced neutrophil transmigration in murine lungs of TLR4 knockout mice. Wild-type and TLR4 knockout mice were intraperitoneally administered with PBS or E. coli OMVs (15 μg in total protein amount) for 6 h. Five animals were used in each group. (A,B) The lung sections of five mice at 6 h after E. coli OMV introduction were immunostained with anti-NIMP-R14 (green; neutrophils) and anti-CD31 (red; endothelial cells) or anti-SP-C (red; lung epithelial cells) antibodies, and counterstained with Hoechst 33258 (blue; nuclei). Representative fluorescence images are shown here. Scale bars = 50 μm. The number of neutrophils per field was counted from five confocal microscopy images obtained from the lung sections of five mice (B) . (C) The concentration of CXCL1 was measured in the BAL fluid by ELISA ( n = 5). Data were represented as mean ± SEM. ∗∗∗ P
    Figure Legend Snippet: Suppression of OMV-induced neutrophil transmigration in murine lungs of TLR4 knockout mice. Wild-type and TLR4 knockout mice were intraperitoneally administered with PBS or E. coli OMVs (15 μg in total protein amount) for 6 h. Five animals were used in each group. (A,B) The lung sections of five mice at 6 h after E. coli OMV introduction were immunostained with anti-NIMP-R14 (green; neutrophils) and anti-CD31 (red; endothelial cells) or anti-SP-C (red; lung epithelial cells) antibodies, and counterstained with Hoechst 33258 (blue; nuclei). Representative fluorescence images are shown here. Scale bars = 50 μm. The number of neutrophils per field was counted from five confocal microscopy images obtained from the lung sections of five mice (B) . (C) The concentration of CXCL1 was measured in the BAL fluid by ELISA ( n = 5). Data were represented as mean ± SEM. ∗∗∗ P

    Techniques Used: Transmigration Assay, Knock-Out, Mouse Assay, Fluorescence, Confocal Microscopy, Concentration Assay, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Inactivation of DNase1L2 and DNase2 in keratinocytes suppresses DNA degradation during epidermal cornification and results in constitutive parakeratosis"

    Article Title: Inactivation of DNase1L2 and DNase2 in keratinocytes suppresses DNA degradation during epidermal cornification and results in constitutive parakeratosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-06652-8

    Immunofluorescence labeling of histones and lamin. Thin section of the skin on the soles of  Dnase1l2 −/−  ( A , C , E ) and  Dnase1l2 −/− Dnase2 Δep  mice ( B , D , F ) were immunolabeled with antibodies against histone H1 ( A , B ), H3 ( C ,  D ), and lamin A/C ( E , F ). The sections were counter-stained with the DNA-specific dye Hoechst 33258. Dotted and dashes lines indicate the outer and the inner border of the stratum corneum, respectively. The data are representative of at least 3 mice per genotype. Scale bars, 20 µm.
    Figure Legend Snippet: Immunofluorescence labeling of histones and lamin. Thin section of the skin on the soles of Dnase1l2 −/− ( A , C , E ) and Dnase1l2 −/− Dnase2 Δep mice ( B , D , F ) were immunolabeled with antibodies against histone H1 ( A , B ), H3 ( C , D ), and lamin A/C ( E , F ). The sections were counter-stained with the DNA-specific dye Hoechst 33258. Dotted and dashes lines indicate the outer and the inner border of the stratum corneum, respectively. The data are representative of at least 3 mice per genotype. Scale bars, 20 µm.

    Techniques Used: Immunofluorescence, Labeling, Mouse Assay, Immunolabeling, Staining

    Isolated corneocytes of DNase1L2/DNase2-deficient mice retain DNA in a nucleus-shaped compartment. Corneocytes were isolated from the soles ( A–D ), ears ( E–H ), and back ( I – L ) of wild-type ( A , B , E,F , I , J ) and  Dnase1l2 −/− Dnase2 Δep  ( C , D , G,H , K , L ) mice. The corneocytes were incubated with DNA-specific dye Hoechst 33258 and fluorescence images in which white signals indicate the labeling (left panels) as well as bright-field images under phase contrast (right panels) were recorded. Scale bars, 20 µm.
    Figure Legend Snippet: Isolated corneocytes of DNase1L2/DNase2-deficient mice retain DNA in a nucleus-shaped compartment. Corneocytes were isolated from the soles ( A–D ), ears ( E–H ), and back ( I – L ) of wild-type ( A , B , E,F , I , J ) and Dnase1l2 −/− Dnase2 Δep ( C , D , G,H , K , L ) mice. The corneocytes were incubated with DNA-specific dye Hoechst 33258 and fluorescence images in which white signals indicate the labeling (left panels) as well as bright-field images under phase contrast (right panels) were recorded. Scale bars, 20 µm.

    Techniques Used: Isolation, Mouse Assay, Incubation, Fluorescence, Labeling

    DNase1L2/DNase2 double knockout causes parakeratosis. Thin section of the skin from  Dnase1l2 −/−  ( A , C , E , G ) and  Dnase1l2 −/− Dnase2 Δep  ( B , D , F , H ) mice were stained with hematoxylin and eosin ( H  and  E ) ( A , B ) and the DNA-specific dye Hoechst 33258 ( C – H ). The skin was prepared from the footpads ( A , D ), ears ( E , F ), and back ( G , H ). Continuous and dashes lines indicate the outer and the inner border of the stratum corneum, respectively. The basal layer of the epidermis is indicated by dotted lines. Arrows indicate nuclear remnants detected in terminally differentiated corneocytes of  Dnase2 ∆ep  mice. The data are representative of at least 3 mice per genotype. Scale bars, 20 µm.
    Figure Legend Snippet: DNase1L2/DNase2 double knockout causes parakeratosis. Thin section of the skin from Dnase1l2 −/− ( A , C , E , G ) and Dnase1l2 −/− Dnase2 Δep ( B , D , F , H ) mice were stained with hematoxylin and eosin ( H and E ) ( A , B ) and the DNA-specific dye Hoechst 33258 ( C – H ). The skin was prepared from the footpads ( A , D ), ears ( E , F ), and back ( G , H ). Continuous and dashes lines indicate the outer and the inner border of the stratum corneum, respectively. The basal layer of the epidermis is indicated by dotted lines. Arrows indicate nuclear remnants detected in terminally differentiated corneocytes of Dnase2 ∆ep mice. The data are representative of at least 3 mice per genotype. Scale bars, 20 µm.

    Techniques Used: Double Knockout, Mouse Assay, Staining

    DNA in DNase1L2/DNase2-deficient corneocytes is partially degraded. Thin section of the skin on the footpads ( A , B ), ears ( C , D ), and back ( E , F ) of  Dnase1l2 −/−  ( A , C , E ) and  Dnase1l2 −/− Dnase2 Δep  ( B , D , F ) mice were subjected to TUNEL labeling and counter-stained with the DNA-specific dye Hoechst 33258. Continuous and dashes lines indicate the outer and the inner border of the stratum corneum, respectively. The basal layer of the epidermis is indicated by dotted lines. The data are representative of at least 3 mice per genotype. Scale bars, 20 µm.
    Figure Legend Snippet: DNA in DNase1L2/DNase2-deficient corneocytes is partially degraded. Thin section of the skin on the footpads ( A , B ), ears ( C , D ), and back ( E , F ) of Dnase1l2 −/− ( A , C , E ) and Dnase1l2 −/− Dnase2 Δep ( B , D , F ) mice were subjected to TUNEL labeling and counter-stained with the DNA-specific dye Hoechst 33258. Continuous and dashes lines indicate the outer and the inner border of the stratum corneum, respectively. The basal layer of the epidermis is indicated by dotted lines. The data are representative of at least 3 mice per genotype. Scale bars, 20 µm.

    Techniques Used: Mouse Assay, TUNEL Assay, Labeling, Staining

    3) Product Images from "Influence of DNA Structure on Adjacent Site Cooperative Binding"

    Article Title: Influence of DNA Structure on Adjacent Site Cooperative Binding

    Journal:

    doi: 10.1021/jp801997v

    Binding curve for the interactions between Hoechst 33258 and CT4GCA4G DNA hairpin at 25 °C in MES 10 buffer. The binding constants were evaluated by fitting the data with a two-site binding model (see Materials and Methods).
    Figure Legend Snippet: Binding curve for the interactions between Hoechst 33258 and CT4GCA4G DNA hairpin at 25 °C in MES 10 buffer. The binding constants were evaluated by fitting the data with a two-site binding model (see Materials and Methods).

    Techniques Used: Binding Assay

    Methyl-H6 portion of TOCSY experiments of Hoechst 33258/DNA hairpin complexes in 0:1, 1:1, and 2:1 ratios at 35 °C with an 80 ms mixing time. Three peaks in the 1:1 complex are not present in 0 and 2 molar ratio. These peaks are shown in red and
    Figure Legend Snippet: Methyl-H6 portion of TOCSY experiments of Hoechst 33258/DNA hairpin complexes in 0:1, 1:1, and 2:1 ratios at 35 °C with an 80 ms mixing time. Three peaks in the 1:1 complex are not present in 0 and 2 molar ratio. These peaks are shown in red and

    Techniques Used: Mass Spectrometry

    SPR sensorgrams for binding of Hoechst 33258 to the CT4GCA4G DNA hairpin at 25 °C in MES 10 buffer. The concentration ranges are 0 (bottom curve) to 80 nM (upper curve).
    Figure Legend Snippet: SPR sensorgrams for binding of Hoechst 33258 to the CT4GCA4G DNA hairpin at 25 °C in MES 10 buffer. The concentration ranges are 0 (bottom curve) to 80 nM (upper curve).

    Techniques Used: SPR Assay, Binding Assay, Concentration Assay

    SPR Analysis of 2:1 Binding of Hoechst 33258 to DNA
    Figure Legend Snippet: SPR Analysis of 2:1 Binding of Hoechst 33258 to DNA

    Techniques Used: SPR Assay, Binding Assay

    Derivative plots of the absorbance at 260 nm with respect to temperature vs temperature for CT4GCA4G DNA duplex (top) and CT4GCA4G DNA hairpin (bottom) in the presence of a series of ratios of Hoechst 33258 in MES 05 buffer.
    Figure Legend Snippet: Derivative plots of the absorbance at 260 nm with respect to temperature vs temperature for CT4GCA4G DNA duplex (top) and CT4GCA4G DNA hairpin (bottom) in the presence of a series of ratios of Hoechst 33258 in MES 05 buffer.

    Techniques Used:

    TOCSY experiments of 0, 1, and 2 molar ratio of Hoechst 33258/DNA duplex in the methyl-H6 region. The temperature is 35 °C. Red peaks are free DNA, and blue peaks are 2:1 ratio. The mixing time is 80 ms.
    Figure Legend Snippet: TOCSY experiments of 0, 1, and 2 molar ratio of Hoechst 33258/DNA duplex in the methyl-H6 region. The temperature is 35 °C. Red peaks are free DNA, and blue peaks are 2:1 ratio. The mixing time is 80 ms.

    Techniques Used: Mass Spectrometry

    4) Product Images from "An Accessory to the 'Trinity': SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses"

    Article Title: An Accessory to the 'Trinity': SR-As Are Essential Pathogen Sensors of Extracellular dsRNA, Mediating Entry and Leading to Subsequent Type I IFN Responses

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000829

    SR-As mediate viral dsRNA binding, entry and resulting ISG induction in MEFs using a mechanism dependent on clathrin-mediated endocytosis. ( A ) Cellular binding of viral dsRNA was observed by fluorescence microscopy in MEFs derived from C57Bl/6 mice treated with either 200 bp or 1000 bp Alexafluor 488 labeled viral dsRNA ( v 200 or  v 1000 respectively, both at 1 µg/mL) for 1h, in the presence or absence of fucoidin or fetuin (both at 100 µg/mL). Cells were fixed and nuclei counterstained with Hoechst 33258. Magnification 400X. ( B ) Fluorescently labeled dsRNA entry was quantified using cells treated with Alexafluor 488 labeled  v 200 or  v 1000 (both at 1 µg/mL) for 1h in the presence of fucoidin or fetuin (both at 100 µg/mL). Cells treated with dsRNA alone (dsRNA) were set at 100% fluorescence. These data include three independent experiments ± SEM. Statistical analysis was performed by a one-way ANOVA with Tukey post test (*** p
    Figure Legend Snippet: SR-As mediate viral dsRNA binding, entry and resulting ISG induction in MEFs using a mechanism dependent on clathrin-mediated endocytosis. ( A ) Cellular binding of viral dsRNA was observed by fluorescence microscopy in MEFs derived from C57Bl/6 mice treated with either 200 bp or 1000 bp Alexafluor 488 labeled viral dsRNA ( v 200 or v 1000 respectively, both at 1 µg/mL) for 1h, in the presence or absence of fucoidin or fetuin (both at 100 µg/mL). Cells were fixed and nuclei counterstained with Hoechst 33258. Magnification 400X. ( B ) Fluorescently labeled dsRNA entry was quantified using cells treated with Alexafluor 488 labeled v 200 or v 1000 (both at 1 µg/mL) for 1h in the presence of fucoidin or fetuin (both at 100 µg/mL). Cells treated with dsRNA alone (dsRNA) were set at 100% fluorescence. These data include three independent experiments ± SEM. Statistical analysis was performed by a one-way ANOVA with Tukey post test (*** p

    Techniques Used: Binding Assay, Fluorescence, Microscopy, Derivative Assay, Mouse Assay, Labeling

    dsRNA binding is dependent on all candidate SR-A family members. DsRNA binding, entry and subsequent induction of ISGs was measured in balb-c derived MEFs treated with media alone (OM), Dharmafect alone (DF), SR-AI/II specific siRNA (I/II) or a combination of SR-AI/II, SCARA3, SCARA4 and SCARA5 siRNA oligomers (-I/II,3,4,5) for 24h. ( A ) Binding of dsRNA was measured in MEFs treated with SR-A siRNA for 24h followed by Alexafluor 488 labeled  v 1000 (1 µg/mL) for 1h. Nuclei were counterstained with Hoechst 33258. Magnification 400X. ( B ) Entry of Alexafluor 488 labeled  v 1000 was measured by fluorescence plate reader, in balb-c MEFs treated with siRNA for 24h followed by 1h treatment with fluorescently labeled  v 1000 (1 µg/mL). Results are reported as a % of DF alone (set to 100%). These data include three independent experiments and are reported as an average ± SEM. Statistical analysis was performed by a one-way ANOVA with a Dunnett's post test, with DF being the control comparison (** p
    Figure Legend Snippet: dsRNA binding is dependent on all candidate SR-A family members. DsRNA binding, entry and subsequent induction of ISGs was measured in balb-c derived MEFs treated with media alone (OM), Dharmafect alone (DF), SR-AI/II specific siRNA (I/II) or a combination of SR-AI/II, SCARA3, SCARA4 and SCARA5 siRNA oligomers (-I/II,3,4,5) for 24h. ( A ) Binding of dsRNA was measured in MEFs treated with SR-A siRNA for 24h followed by Alexafluor 488 labeled v 1000 (1 µg/mL) for 1h. Nuclei were counterstained with Hoechst 33258. Magnification 400X. ( B ) Entry of Alexafluor 488 labeled v 1000 was measured by fluorescence plate reader, in balb-c MEFs treated with siRNA for 24h followed by 1h treatment with fluorescently labeled v 1000 (1 µg/mL). Results are reported as a % of DF alone (set to 100%). These data include three independent experiments and are reported as an average ± SEM. Statistical analysis was performed by a one-way ANOVA with a Dunnett's post test, with DF being the control comparison (** p

    Techniques Used: Binding Assay, Derivative Assay, Labeling, Fluorescence

    5) Product Images from "Impact of neonatal hypoxia‐ischaemia on oligodendrocyte survival, maturation and myelinating potential"

    Article Title: Impact of neonatal hypoxia‐ischaemia on oligodendrocyte survival, maturation and myelinating potential

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13309

    Effect of glucose and oxygen deprivation on the survival, proliferation and differentiation of rat oligodendrocyte progenitors within organotypic hippocampal slices during 7  DIV . Anti‐ PLP  staining (red) of mature oligodendrocytes: Colocalization with gelatinases (green) was observed neither in control slices ( A – C ) nor in slices injured by  OGD  procedure ( D – F ). Cell nuclei (blue) are visualized with Hoechst 33258. Scale bar is the equivalent of 100 μm. ( G ) Statistical analysis of oligodendrocyte proliferation and maturation in slices subjected to  OGD  procedure; ( H ) statistical analysis of the amount of the  OPC s which are newly born (BrdU + ) after the insult and progress in their maturation. The calculated differences were marked as significant if: ** P
    Figure Legend Snippet: Effect of glucose and oxygen deprivation on the survival, proliferation and differentiation of rat oligodendrocyte progenitors within organotypic hippocampal slices during 7 DIV . Anti‐ PLP staining (red) of mature oligodendrocytes: Colocalization with gelatinases (green) was observed neither in control slices ( A – C ) nor in slices injured by OGD procedure ( D – F ). Cell nuclei (blue) are visualized with Hoechst 33258. Scale bar is the equivalent of 100 μm. ( G ) Statistical analysis of oligodendrocyte proliferation and maturation in slices subjected to OGD procedure; ( H ) statistical analysis of the amount of the OPC s which are newly born (BrdU + ) after the insult and progress in their maturation. The calculated differences were marked as significant if: ** P

    Techniques Used: Plasmid Purification, Staining, IF-P

    Amounts of major myelin components in both the organotypic hippocampal slices and in the rat brains 7 days after hypoxic‐ischaemic incident; ( A – C ) mature, control  MBP ‐positive (red) oligodendrocytes in organotypic hippocampal slices, solely (white arrows) expressing gelatinases (green). ( D – F ) enlargement of the white frame to show branched oligodendrocyte morphology; ( G – I )  OGD ‐subjected slices; ( J – L ) magnification of the previous picture. Cell nuclei are stained with Hoechst 33258 (blue); Scale bar corresponds to 100 μm. ( M ) Quantitative analysis of  MBP  and  PLP  amounts in organotypic hippocampal slices; ( N ) quantitative analysis of  MBP  and  PLP  amounts in rat brains. The calculated differences were considered as significant if * P
    Figure Legend Snippet: Amounts of major myelin components in both the organotypic hippocampal slices and in the rat brains 7 days after hypoxic‐ischaemic incident; ( A – C ) mature, control MBP ‐positive (red) oligodendrocytes in organotypic hippocampal slices, solely (white arrows) expressing gelatinases (green). ( D – F ) enlargement of the white frame to show branched oligodendrocyte morphology; ( G – I ) OGD ‐subjected slices; ( J – L ) magnification of the previous picture. Cell nuclei are stained with Hoechst 33258 (blue); Scale bar corresponds to 100 μm. ( M ) Quantitative analysis of MBP and PLP amounts in organotypic hippocampal slices; ( N ) quantitative analysis of MBP and PLP amounts in rat brains. The calculated differences were considered as significant if * P

    Techniques Used: Expressing, Staining, Plasmid Purification, IF-P

    Effect of glucose and oxygen deprivation on the survival and proliferation of oligodendrocyte progenitors within organotypic hippocampal slices: ( A ) live image of control hippocampal slice; ( B )  OGD ‐subjected slice with visible disintegration of its characteristic morphology; ( C )  NG 2‐positive (red) progenitors in controls; ( D ) reduced number of  NG 2 +  (red) progenitors after  OGD  insult; ( E ) oligodendrocyte progenitors ( NG 2 + , red) expressing gelatinases ( MMP s, green) in controls; ( F ) colocalization of  NG 2 marker (red) and gelatinase activity (green) in injured slices; ( G ) proliferating, Ki67 +  (red) progenitors ( NG 2 + , green) in control slices (white arrows indicate double‐labelled cells); ( H ) colocalization (white arrows) of  OPC s ( NG 2, green) and marker of dividing cells‐Ki67 (red) 7 days after  OGD , cell nuclei are stained with Hoechst 33258 (blue); ( I ) newly generated (BrdU + , green)  OPC  ( NG 2 + , red) in control slices; ( J )  OPC s ( NG 2 + , red) generated (BrdU + , green) after  OGD  insult; ( K ) immature (O4‐positive, red) BrdU +  (green) oligodendrocytes in controls; ( L ) double‐labelled immature oligodendrocytes (O4 + , red), born‐as indicated by BrdU +  (green) incorporation‐after  OGD  procedure. Scale bar is equivalent of 50 μm.
    Figure Legend Snippet: Effect of glucose and oxygen deprivation on the survival and proliferation of oligodendrocyte progenitors within organotypic hippocampal slices: ( A ) live image of control hippocampal slice; ( B ) OGD ‐subjected slice with visible disintegration of its characteristic morphology; ( C ) NG 2‐positive (red) progenitors in controls; ( D ) reduced number of NG 2 + (red) progenitors after OGD insult; ( E ) oligodendrocyte progenitors ( NG 2 + , red) expressing gelatinases ( MMP s, green) in controls; ( F ) colocalization of NG 2 marker (red) and gelatinase activity (green) in injured slices; ( G ) proliferating, Ki67 + (red) progenitors ( NG 2 + , green) in control slices (white arrows indicate double‐labelled cells); ( H ) colocalization (white arrows) of OPC s ( NG 2, green) and marker of dividing cells‐Ki67 (red) 7 days after OGD , cell nuclei are stained with Hoechst 33258 (blue); ( I ) newly generated (BrdU + , green) OPC ( NG 2 + , red) in control slices; ( J ) OPC s ( NG 2 + , red) generated (BrdU + , green) after OGD insult; ( K ) immature (O4‐positive, red) BrdU + (green) oligodendrocytes in controls; ( L ) double‐labelled immature oligodendrocytes (O4 + , red), born‐as indicated by BrdU + (green) incorporation‐after OGD procedure. Scale bar is equivalent of 50 μm.

    Techniques Used: Expressing, Marker, Activity Assay, Staining, Generated

    6) Product Images from "APTM, a Thiophene Heterocyclic Compound, Inhibits Human Colon Cancer HCT116 Cell Proliferation Through p53-Dependent Induction of Apoptosis"

    Article Title: APTM, a Thiophene Heterocyclic Compound, Inhibits Human Colon Cancer HCT116 Cell Proliferation Through p53-Dependent Induction of Apoptosis

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2017.3962

    Depletion of p53 attenuates APTM effect on apoptosis and cell proliferation. HCT116  p53 −/−  cells were treated with APTM at indicated concentrations for 48 h.  (A)  Visualization of apoptotic morphological changes by a fluorescent microscope with Hoechst 33258 staining. Representative pictures are shown (400 × ).  (B)  Cells were stained with annexin V-FITC/PI and apoptosis tested by flow cytometry. Representative contour diagrams are shown.  (C)  Western blotting analysis of apoptosis proteins.  (D)  Cell growth curve. The cell number at each dose is presented as the percentage of control. Average values are from three independent experiments performed in duplicate ( n  = 3). Data are shown as mean ± SD.
    Figure Legend Snippet: Depletion of p53 attenuates APTM effect on apoptosis and cell proliferation. HCT116 p53 −/− cells were treated with APTM at indicated concentrations for 48 h. (A) Visualization of apoptotic morphological changes by a fluorescent microscope with Hoechst 33258 staining. Representative pictures are shown (400 × ). (B) Cells were stained with annexin V-FITC/PI and apoptosis tested by flow cytometry. Representative contour diagrams are shown. (C) Western blotting analysis of apoptosis proteins. (D) Cell growth curve. The cell number at each dose is presented as the percentage of control. Average values are from three independent experiments performed in duplicate ( n  = 3). Data are shown as mean ± SD.

    Techniques Used: Microscopy, Staining, Flow Cytometry, Cytometry, Western Blot

    APTM induces apoptosis in HCT116 cells. HCT116 cells were treated with APTM at indicated concentrations for 48 h.  (A)  Visualization of apoptotic morphological changes by a fluorescent microscope with Hoechst 33258 staining. Representative pictures are shown (400 × ).  (B)  Cells were stained with annexin V-FITC/PI and apoptosis tested by flow cytometry. Representative contour diagrams are shown ( left ). Fractions of apoptotic cells were quantified ( right ). Average values are from three independent experiments ( n  = 3).  (C)  Western blotting analysis of apoptosis and survival-related proteins ( left ). Band intensity of Bax and Bcl-2 proteins was determined and expressed as the Bax/Bcl-2 ratio ( right ). Data are shown as mean ± SD. PI, propidium iodide.
    Figure Legend Snippet: APTM induces apoptosis in HCT116 cells. HCT116 cells were treated with APTM at indicated concentrations for 48 h. (A) Visualization of apoptotic morphological changes by a fluorescent microscope with Hoechst 33258 staining. Representative pictures are shown (400 × ). (B) Cells were stained with annexin V-FITC/PI and apoptosis tested by flow cytometry. Representative contour diagrams are shown ( left ). Fractions of apoptotic cells were quantified ( right ). Average values are from three independent experiments ( n  = 3). (C) Western blotting analysis of apoptosis and survival-related proteins ( left ). Band intensity of Bax and Bcl-2 proteins was determined and expressed as the Bax/Bcl-2 ratio ( right ). Data are shown as mean ± SD. PI, propidium iodide.

    Techniques Used: Microscopy, Staining, Flow Cytometry, Cytometry, Western Blot

    7) Product Images from "Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors"

    Article Title: Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2018.203

    ADSC coculturing increased levels of bFGF and SCF. (A, B) Real-time PCR (A) and Western blot analysis (B) results for relative ratios of bFGF and SCF mRNA and protein levels in cell lysates. (C) ELISA results for concentrations of bFGF and SCF proteins in culture supernatants. (D) Representative immunofluorescent staining using anti-bFGF or anti-SCF antibody. All these studies were performed using ADSC monocultures (ADSC), primary cultured normal adult human melanocyte monocultures (MC), and MC+ADSC cocultures (MC+ADSC). GAPDH and α-tubulin were used as internal controls for real-time PCR and Western blot analysis, respectively. In immunofluorescence staining, nuclei were counter-stained with Hoechst 33258 (Bar=0.1 mm) and intensities in randomly selected total 20 ADSCs and/or melanocytes were measured using Wright Cell Imaging Facility ImageJ software and compared between monocultures and cocultures. Data in each graph represent mean ± SD of three or four independent experiments. * p
    Figure Legend Snippet: ADSC coculturing increased levels of bFGF and SCF. (A, B) Real-time PCR (A) and Western blot analysis (B) results for relative ratios of bFGF and SCF mRNA and protein levels in cell lysates. (C) ELISA results for concentrations of bFGF and SCF proteins in culture supernatants. (D) Representative immunofluorescent staining using anti-bFGF or anti-SCF antibody. All these studies were performed using ADSC monocultures (ADSC), primary cultured normal adult human melanocyte monocultures (MC), and MC+ADSC cocultures (MC+ADSC). GAPDH and α-tubulin were used as internal controls for real-time PCR and Western blot analysis, respectively. In immunofluorescence staining, nuclei were counter-stained with Hoechst 33258 (Bar=0.1 mm) and intensities in randomly selected total 20 ADSCs and/or melanocytes were measured using Wright Cell Imaging Facility ImageJ software and compared between monocultures and cocultures. Data in each graph represent mean ± SD of three or four independent experiments. * p

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Cell Culture, Immunofluorescence, Imaging, Software

    Upregulation of bFGF and SCF by ADSCs was involved in integrin-mediated melanocyte adhesion to ECM. (A, B) Western blot analysis for relative levels of integrins β1, α5, and α6 proteins in melanocytes treated with or without anti-bFGF (A) or anti-SCF antibody (B). (C, D) Adhesion assay using fibronectin- (C) or laminin-coated culture dishes (D) treated with or without anti-bFGF or anti-SCF antibody. Nuclei were stained with Hoechst 33258 (Bar=0.2 mm). These experiments were performed using melanocyte monocultures (monoMC) and melanocytes taken from upper insert after coculturing with ADSCs in lower chamber (coMC). Data in each graph represent mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Upregulation of bFGF and SCF by ADSCs was involved in integrin-mediated melanocyte adhesion to ECM. (A, B) Western blot analysis for relative levels of integrins β1, α5, and α6 proteins in melanocytes treated with or without anti-bFGF (A) or anti-SCF antibody (B). (C, D) Adhesion assay using fibronectin- (C) or laminin-coated culture dishes (D) treated with or without anti-bFGF or anti-SCF antibody. Nuclei were stained with Hoechst 33258 (Bar=0.2 mm). These experiments were performed using melanocyte monocultures (monoMC) and melanocytes taken from upper insert after coculturing with ADSCs in lower chamber (coMC). Data in each graph represent mean ± SD of three independent experiments. * p

    Techniques Used: Western Blot, Cell Adhesion Assay, Staining

    Grafting of melanocyte-ADSC cocultures increased levels of growth factors and integrins in nude mice. Representative immunofluorescent staining using anti-bFGF or anti-SCF (A) and anti-integrin β1, anti-integrin α5, or anti-integrin α6 (B) in skin specimens of nude mice grafted with ADSC monocultures (ADSC), melanocyte monocultures (MC), or melanocyte+ADSC cocultures (MC+ADSC). Nuclei were count-stained with Hoechst 33258 (Bar=0.05 mm). Intensities were measured in five randomly selected high-power fields (x400) of the dermis using Wright Cell Imaging Facility ImageJ software. Data in each graph represent mean ± SD from five nude mice. * p
    Figure Legend Snippet: Grafting of melanocyte-ADSC cocultures increased levels of growth factors and integrins in nude mice. Representative immunofluorescent staining using anti-bFGF or anti-SCF (A) and anti-integrin β1, anti-integrin α5, or anti-integrin α6 (B) in skin specimens of nude mice grafted with ADSC monocultures (ADSC), melanocyte monocultures (MC), or melanocyte+ADSC cocultures (MC+ADSC). Nuclei were count-stained with Hoechst 33258 (Bar=0.05 mm). Intensities were measured in five randomly selected high-power fields (x400) of the dermis using Wright Cell Imaging Facility ImageJ software. Data in each graph represent mean ± SD from five nude mice. * p

    Techniques Used: Mouse Assay, Staining, Imaging, Software

    ADSC coculturing enhanced melanocyte adhesion to ECM through β1 integrin upregulation. (A) Western blot analysis for relative levels of integrins β1, α5, and α6 proteins. α-tubulin was used as an internal control. (B) Representative immunofluorescent staining using anti-integrin β1, anti-integrin α5, or anti-integrin α6 antibody. Nuclei were count-stained with Hoechst 33258 (Bar=0.05 mm) and intensities were measured using Wright Cell Imaging Facility ImageJ software. (C, D) Adhesion assay on fibronectin- (C) or laminin-coated culture dishes (D) treated with or without anti-integrin β1 antibody. Nuclei were stained with Hoechst 33258 (Bar=0.2 mm). These studies were done using melanocyte monocultures (monoMC) and melanocytes taken from upper insert after coculturing with ADSCs in lower chamber (coMC). Data in each graph represent mean ± SD of three independent experiments. * p
    Figure Legend Snippet: ADSC coculturing enhanced melanocyte adhesion to ECM through β1 integrin upregulation. (A) Western blot analysis for relative levels of integrins β1, α5, and α6 proteins. α-tubulin was used as an internal control. (B) Representative immunofluorescent staining using anti-integrin β1, anti-integrin α5, or anti-integrin α6 antibody. Nuclei were count-stained with Hoechst 33258 (Bar=0.05 mm) and intensities were measured using Wright Cell Imaging Facility ImageJ software. (C, D) Adhesion assay on fibronectin- (C) or laminin-coated culture dishes (D) treated with or without anti-integrin β1 antibody. Nuclei were stained with Hoechst 33258 (Bar=0.2 mm). These studies were done using melanocyte monocultures (monoMC) and melanocytes taken from upper insert after coculturing with ADSCs in lower chamber (coMC). Data in each graph represent mean ± SD of three independent experiments. * p

    Techniques Used: Western Blot, Staining, Imaging, Software, Cell Adhesion Assay

    8) Product Images from "MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2"

    Article Title: MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0149965

    MLIF protects SH-SY5Y cells against apoptosis in an eEF1A2-dependent manner. OGD-exposed SH-SY5Y cells transfected with eEF1A2 siRNA or negative control siRNA (NC) were treated with MLIF (0.1 μg/ml). Cell survival was measured using MTT assay (A). Representative annexin V/PI labeling, assessed by flow cytometry, was used to analyze the ratio of apoptotic SH-SY5Y cells (B, D). Hoechst 33258 staining was used to evaluate the nuclear morphology of SH-SY5Y cells (C, E). Data are expressed as the mean ± SEM. Results were analyzed using one-way ANOVA; n = 3. ** P
    Figure Legend Snippet: MLIF protects SH-SY5Y cells against apoptosis in an eEF1A2-dependent manner. OGD-exposed SH-SY5Y cells transfected with eEF1A2 siRNA or negative control siRNA (NC) were treated with MLIF (0.1 μg/ml). Cell survival was measured using MTT assay (A). Representative annexin V/PI labeling, assessed by flow cytometry, was used to analyze the ratio of apoptotic SH-SY5Y cells (B, D). Hoechst 33258 staining was used to evaluate the nuclear morphology of SH-SY5Y cells (C, E). Data are expressed as the mean ± SEM. Results were analyzed using one-way ANOVA; n = 3. ** P

    Techniques Used: Transfection, Negative Control, MTT Assay, Labeling, Flow Cytometry, Cytometry, Staining

    The effect of MLIF on OGD-induced SH-SY5Y neuroblastoma injury was evaluated using MTT assay, flow cytometry and Hoechst staining assay. SH-SY5Y cells were exposed to OGD for 6h. After treatment with MLIF (0.1, 1.0, 10 μg/ml), MTT assay was used to measure the cell survival ratio (A). Annexin V/PI labeling, assessed by flow cytometry (B, D), and Hoechst 33258 staining (C, E) were performed to evaluate apoptosis in SH-SY5Y cells. Data are expressed as the mean ± SEM. Results were analyzed with one-way ANOVA; n = 3. ** P
    Figure Legend Snippet: The effect of MLIF on OGD-induced SH-SY5Y neuroblastoma injury was evaluated using MTT assay, flow cytometry and Hoechst staining assay. SH-SY5Y cells were exposed to OGD for 6h. After treatment with MLIF (0.1, 1.0, 10 μg/ml), MTT assay was used to measure the cell survival ratio (A). Annexin V/PI labeling, assessed by flow cytometry (B, D), and Hoechst 33258 staining (C, E) were performed to evaluate apoptosis in SH-SY5Y cells. Data are expressed as the mean ± SEM. Results were analyzed with one-way ANOVA; n = 3. ** P

    Techniques Used: MTT Assay, Flow Cytometry, Cytometry, Staining, Labeling

    9) Product Images from "The Neuroprotective Effect Exerted by Oligodendroglial Progenitors on Ischemically Impaired Hippocampal Cells"

    Article Title: The Neuroprotective Effect Exerted by Oligodendroglial Progenitors on Ischemically Impaired Hippocampal Cells

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-013-8549-9

    The effect of secreted BDNF on cultured hippocampal slices.  a  Neutralization of BDNF diminished the number of NF200-positive neurons, abolishing the neuroprotective role of this factor. Immunostaining of hippocampal organotypic slices: neuronal marker NF200 ( green ), dead cell indicator PI ( red ), cell nuclei tracker Hoechst 33258 ( blue ).Scale bar =200 μm.  b  The measurement of BDNF concentration in control hippocampal slices ( Hcontr ), hippocampal slices subjected to OGD procedure ( HOGD ), oligodendroglial progenitors ( OPC ) and mature oligodendrocytes ( OLS ). The statistical differences of the data analyzed is  p
    Figure Legend Snippet: The effect of secreted BDNF on cultured hippocampal slices. a Neutralization of BDNF diminished the number of NF200-positive neurons, abolishing the neuroprotective role of this factor. Immunostaining of hippocampal organotypic slices: neuronal marker NF200 ( green ), dead cell indicator PI ( red ), cell nuclei tracker Hoechst 33258 ( blue ).Scale bar =200 μm. b The measurement of BDNF concentration in control hippocampal slices ( Hcontr ), hippocampal slices subjected to OGD procedure ( HOGD ), oligodendroglial progenitors ( OPC ) and mature oligodendrocytes ( OLS ). The statistical differences of the data analyzed is p

    Techniques Used: Cell Culture, Neutralization, Immunostaining, Marker, Concentration Assay

    Adherent dividing population of OPCs used for co-culturing with organotypic hippocampal slices.  a  Phase contrast microscopy of freshly isolated and purified OPCs.  b  Oligodendrocyte progenitors 6 h after seeding on uncoated glass cover slips: specific immunodetection of NG2 ( green ) and CNP ( red ) antigens.  c ,  d  Dividing OPCs cultured for 24 h: NG2 ( green ) and Ki67 ( red ) markers. Cell nuclei ( blue ) are stained with Hoechst 33258. Scale bar = 50 μm
    Figure Legend Snippet: Adherent dividing population of OPCs used for co-culturing with organotypic hippocampal slices. a Phase contrast microscopy of freshly isolated and purified OPCs. b Oligodendrocyte progenitors 6 h after seeding on uncoated glass cover slips: specific immunodetection of NG2 ( green ) and CNP ( red ) antigens. c , d Dividing OPCs cultured for 24 h: NG2 ( green ) and Ki67 ( red ) markers. Cell nuclei ( blue ) are stained with Hoechst 33258. Scale bar = 50 μm

    Techniques Used: Microscopy, Isolation, Purification, Immunodetection, Cell Culture, Staining

    Quantity of OPC population during 1-week co-culture with hippocampal slices subjected to the OGD injury:  a – i  phase contrast and NG2 ( green ), Ki67 ( red ) and Hoechst 33258 ( blue ) immunostaining of dividing and differentiating cells.  a – c  Freshly isolated and seeded NG2 +  cells during first 24 h in vitro. Single, dividing cells with a few projections are present.  d – f  After 48 h in vitro, cells are characterized by still high proliferation rate and by more complex morphology.  g – i  In developing oligodendrocyte fraction, in 72 h in vitro, NG2-positive cells still predominate, are able to divide and elaborate highly branched processes. Scale bar =50 μm.  j  Gradual decline in OPCs population during 7 DIV
    Figure Legend Snippet: Quantity of OPC population during 1-week co-culture with hippocampal slices subjected to the OGD injury: a – i phase contrast and NG2 ( green ), Ki67 ( red ) and Hoechst 33258 ( blue ) immunostaining of dividing and differentiating cells. a – c Freshly isolated and seeded NG2 + cells during first 24 h in vitro. Single, dividing cells with a few projections are present. d – f After 48 h in vitro, cells are characterized by still high proliferation rate and by more complex morphology. g – i In developing oligodendrocyte fraction, in 72 h in vitro, NG2-positive cells still predominate, are able to divide and elaborate highly branched processes. Scale bar =50 μm. j Gradual decline in OPCs population during 7 DIV

    Techniques Used: Co-Culture Assay, Immunostaining, Isolation, In Vitro

    10) Product Images from "The Differentiation of Rat Oligodendroglial Cells Is Highly Influenced by the Oxygen Tension: In Vitro Model Mimicking Physiologically Normoxic Conditions"

    Article Title: The Differentiation of Rat Oligodendroglial Cells Is Highly Influenced by the Oxygen Tension: In Vitro Model Mimicking Physiologically Normoxic Conditions

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020331

    The influence of distinct oxygen conditions on oligodendrocyte proliferation and differentiation. ( A ) The CNP-positive oligodendrocytes ( green ) visible in hippocampal slices after 7 DIV; The cell nuclei were labelled with Hoechst 33258 ( blue ); ( B ) The diagram shows the total number of OPCs, dividing progenitors, and differentiating oligodendrocytes in hippocampal slices cultured in distinct oxygen concentrations. The scale bar is equivalent to 20 µm. The calculated differences were regarded as statistically significant if *  p
    Figure Legend Snippet: The influence of distinct oxygen conditions on oligodendrocyte proliferation and differentiation. ( A ) The CNP-positive oligodendrocytes ( green ) visible in hippocampal slices after 7 DIV; The cell nuclei were labelled with Hoechst 33258 ( blue ); ( B ) The diagram shows the total number of OPCs, dividing progenitors, and differentiating oligodendrocytes in hippocampal slices cultured in distinct oxygen concentrations. The scale bar is equivalent to 20 µm. The calculated differences were regarded as statistically significant if * p

    Techniques Used: Cell Culture, IF-P

    Quantification of immunolabeled oligodendrocyte progenitors in different cell culture conditions. Cell cultures were fixed either after 2 or 5 days in vitro. Blue bars show cells maintained in physiological normoxia (5% O 2 ) and green bars represent cells kept at the standard oxygen level (21% O 2 ). ( A ) Expression of the lineage-specific transcription factor Olig1 ( green ), which is detected in the cell cytoplasm on the 2nd DIV (white arrows) and gradually decreases during cell maturation; ( B ) Expression of the transcription factor Olig2 ( green ), which is relevant for the early stages of oligodendrocyte differentiation (white arrows); ( C ) Progenitor cells recognized by the presence on their surface of the NG2 marker; ( D ) Proliferating cells characterized by the presence of the Ki67 marker in the cell nuclei. The cell nuclei were visualized with Hoechst 33258 solution ( blue ). The scale bar is equivalent to 20 µm. The calculated differences were marked as the significant if: *  p
    Figure Legend Snippet: Quantification of immunolabeled oligodendrocyte progenitors in different cell culture conditions. Cell cultures were fixed either after 2 or 5 days in vitro. Blue bars show cells maintained in physiological normoxia (5% O 2 ) and green bars represent cells kept at the standard oxygen level (21% O 2 ). ( A ) Expression of the lineage-specific transcription factor Olig1 ( green ), which is detected in the cell cytoplasm on the 2nd DIV (white arrows) and gradually decreases during cell maturation; ( B ) Expression of the transcription factor Olig2 ( green ), which is relevant for the early stages of oligodendrocyte differentiation (white arrows); ( C ) Progenitor cells recognized by the presence on their surface of the NG2 marker; ( D ) Proliferating cells characterized by the presence of the Ki67 marker in the cell nuclei. The cell nuclei were visualized with Hoechst 33258 solution ( blue ). The scale bar is equivalent to 20 µm. The calculated differences were marked as the significant if: * p

    Techniques Used: Immunolabeling, Cell Culture, In Vitro, Expressing, Marker, IF-P

    The influence of the cell seeding density on OPC proliferation (revealed by Ki67 immunostaining,  green ) and differentiation (estimated by GalC expression,  green ) determined after culturing the cells for 48 h in serum-free conditions in physiological normoxia. ( A ) Cells seeded at a high density (5 × 10 4 /cm 2 ) divide approximately five-fold more frequently than those cultured in low density (1.5 × 10 4 /cm 2 ), as indicated by Ki67 presence in the cell nuclei; ( B ) cell differentiation, verified by the presence of GalC +  oligodendrocytes, is highly influenced by the cell culture density. When cultured in low density, GalC +  cells are significantly more numerous and they are characterized by a much more complex, branched morphology. The cell nuclei were labelled with Hoechst 33258 ( blue ). The scale bar is the equivalent to 100 µm. The calculated differences were considered statistically significant when **  p
    Figure Legend Snippet: The influence of the cell seeding density on OPC proliferation (revealed by Ki67 immunostaining, green ) and differentiation (estimated by GalC expression, green ) determined after culturing the cells for 48 h in serum-free conditions in physiological normoxia. ( A ) Cells seeded at a high density (5 × 10 4 /cm 2 ) divide approximately five-fold more frequently than those cultured in low density (1.5 × 10 4 /cm 2 ), as indicated by Ki67 presence in the cell nuclei; ( B ) cell differentiation, verified by the presence of GalC + oligodendrocytes, is highly influenced by the cell culture density. When cultured in low density, GalC + cells are significantly more numerous and they are characterized by a much more complex, branched morphology. The cell nuclei were labelled with Hoechst 33258 ( blue ). The scale bar is the equivalent to 100 µm. The calculated differences were considered statistically significant when ** p

    Techniques Used: Immunostaining, Expressing, Cell Culture, Cell Differentiation

    Quantification of maturating oligodendrocytes maintained in different culture conditions for either 2 or 5 DIV. The cells were cultured either in 5% ( blue bars ) or 21% O 2  ( green bars) . ( A ) Differentiating, multiprocessed cells are characterized by the presence of the GalC antigen on the cell surface; ( B ) Expression of CNP (2′,3′-Cyclic-nucleotide 3′-phosphodiesterase), the characteristic marker of the oligodendroglial lineage; ( C ) Detection of myelin basic proteins (MPB) in differentiated oligodendrocytes with branched cell processes; ( D ) Presence of proteolipid protein (PLP) in mature cells. The cell nuclei were labelled with Hoechst 33258 ( blue ). The scale bar corresponds to 20 µm. The calculated differences were considered statistically significant if: *  p
    Figure Legend Snippet: Quantification of maturating oligodendrocytes maintained in different culture conditions for either 2 or 5 DIV. The cells were cultured either in 5% ( blue bars ) or 21% O 2 ( green bars) . ( A ) Differentiating, multiprocessed cells are characterized by the presence of the GalC antigen on the cell surface; ( B ) Expression of CNP (2′,3′-Cyclic-nucleotide 3′-phosphodiesterase), the characteristic marker of the oligodendroglial lineage; ( C ) Detection of myelin basic proteins (MPB) in differentiated oligodendrocytes with branched cell processes; ( D ) Presence of proteolipid protein (PLP) in mature cells. The cell nuclei were labelled with Hoechst 33258 ( blue ). The scale bar corresponds to 20 µm. The calculated differences were considered statistically significant if: * p

    Techniques Used: Cell Culture, Expressing, Marker, Plasmid Purification, IF-P

    11) Product Images from "Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins"

    Article Title: Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094383

    A great variety of proteins are detected by antibody, GFP-/EYFP-/RFP-fusion constructs, or X-Gal staining for active β-galactosidase produced by  lacZ -containing  P -element insertion stocks. We consistently used 9–10 hr old prepupal salivary glands for these types of detection. ( a ) Salivary gland showing the presence of nuclear receptor E75 (red) and a portion of the cytoplasmic signaling protein Ras2 (green) in the lumen. The cortical membrane is stained with AF 488 -phalloidin for F-actin. ( b ) Similarly to ( a ), two cytoplasmic proteins, Oho-31 (green) and tight junction membrane protein Arm (red) were found secreted into the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). ( c ) Tumor suppressor protein p127, the product of  l(2)gl  gene (green), and the nucleolar component fibrillarin (red) are found secreted in the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). Fluorescently-tagged constructs (most using GFP-), showed that many fusion proteins were secreted into the lumen. These are exemplified by GFP-Rbp1 ( d ). Examples of proteins monitored via  lacZ -fusion include the transcription factor Ttk ( e ), the dual-specific LAMMER kinase Doa ( f ), the D subunit of the vacuolar H +  vATPase Vha36-1 ( g ) and the transcription factor Fkh ( h ).
    Figure Legend Snippet: A great variety of proteins are detected by antibody, GFP-/EYFP-/RFP-fusion constructs, or X-Gal staining for active β-galactosidase produced by lacZ -containing P -element insertion stocks. We consistently used 9–10 hr old prepupal salivary glands for these types of detection. ( a ) Salivary gland showing the presence of nuclear receptor E75 (red) and a portion of the cytoplasmic signaling protein Ras2 (green) in the lumen. The cortical membrane is stained with AF 488 -phalloidin for F-actin. ( b ) Similarly to ( a ), two cytoplasmic proteins, Oho-31 (green) and tight junction membrane protein Arm (red) were found secreted into the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). ( c ) Tumor suppressor protein p127, the product of l(2)gl gene (green), and the nucleolar component fibrillarin (red) are found secreted in the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). Fluorescently-tagged constructs (most using GFP-), showed that many fusion proteins were secreted into the lumen. These are exemplified by GFP-Rbp1 ( d ). Examples of proteins monitored via lacZ -fusion include the transcription factor Ttk ( e ), the dual-specific LAMMER kinase Doa ( f ), the D subunit of the vacuolar H + vATPase Vha36-1 ( g ) and the transcription factor Fkh ( h ).

    Techniques Used: Construct, Staining, Produced

    Evidence for apocrine secretion of undegraded proteins and the presence of intact genomic DNA in nuclei, and for the release of mitochondria into lumen. Panels a and b show western blots of secreted proteins isolated from the lumen. ( a ) Rab11 protein was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion (lane 3). ( b ) The transcription factor BR-C Z1 was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion from +9–10 hr APF (lane 3). ( c ) In +8–8.5 hr APF prepupae, ribosomal protein Rp40 (green) and β-tubulin (red) are detectable in the lumen of the salivary glands, while the signal for DNA remains nuclear. ( d ) In +9 hr APF prepupae, the ribosomal protein Rp21 (green) and transcription factor E74 (red) are detected in the lumen, while the signal for DNA remains nuclear. ( e ) In +10 hr APF prepupae, both the ribosomal protein p127 (green) and the transcription factor BR-C (red) are detected in the lumen, while the signal for DNA remains nuclear throughout the entire salivary gland, including its columnar, transitional and corpuscular cells; confocal images 80×. ( f ,  g ) Mitochondria are released by apocrine secretion into the lumen as evidenced by chasing a vital Rhodamine 123 signal. In larval as well as early prepupal salivary glands, intact living mitochondria are visible only inside cells ( f ), whereas in +8–10 hr APF prepupae they also can be detected inside the lumen ( g ); both confocal images 630×. This is also consistent with detection of more than dozen of various mitochondrial proteins listed in   Tables 1  through   4 . In addition,  in situ  hybridization with a mitochondrial genome-specific DNA probe (3'-OH end of mt cytochrome c oxidase I, entire coding sequence of mt tRNA-Leu, and 5'-OH end of mt cytochrome c oxidase II) confirmed the presence of mitochondrial DNA in the secretory material in +9 hr APF prepupae ( h ,  i , (green)) along with F-actin ( h ,  j , (blue)). Although nuclear proteins are released by an apocrine mechanism into the lumen, nuclear DNA was never detected in the secretion. When  in situ  hybridization was performed in +9 hr APF prepupae with a probe for a nuclear gene  Doa  locus, signal was found only in nuclei ( k ,  n , (red)) together with Hoechst 33258 staining DNA ( k ,  l , (green)), while F-actin was detectable in the lumen ( k ,  m , (blue)). Remaining confocal images 400×. L in ( f ), ( g ), ( h ) and ( k )  =  lumen.
    Figure Legend Snippet: Evidence for apocrine secretion of undegraded proteins and the presence of intact genomic DNA in nuclei, and for the release of mitochondria into lumen. Panels a and b show western blots of secreted proteins isolated from the lumen. ( a ) Rab11 protein was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion (lane 3). ( b ) The transcription factor BR-C Z1 was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion from +9–10 hr APF (lane 3). ( c ) In +8–8.5 hr APF prepupae, ribosomal protein Rp40 (green) and β-tubulin (red) are detectable in the lumen of the salivary glands, while the signal for DNA remains nuclear. ( d ) In +9 hr APF prepupae, the ribosomal protein Rp21 (green) and transcription factor E74 (red) are detected in the lumen, while the signal for DNA remains nuclear. ( e ) In +10 hr APF prepupae, both the ribosomal protein p127 (green) and the transcription factor BR-C (red) are detected in the lumen, while the signal for DNA remains nuclear throughout the entire salivary gland, including its columnar, transitional and corpuscular cells; confocal images 80×. ( f , g ) Mitochondria are released by apocrine secretion into the lumen as evidenced by chasing a vital Rhodamine 123 signal. In larval as well as early prepupal salivary glands, intact living mitochondria are visible only inside cells ( f ), whereas in +8–10 hr APF prepupae they also can be detected inside the lumen ( g ); both confocal images 630×. This is also consistent with detection of more than dozen of various mitochondrial proteins listed in Tables 1 through 4 . In addition, in situ hybridization with a mitochondrial genome-specific DNA probe (3'-OH end of mt cytochrome c oxidase I, entire coding sequence of mt tRNA-Leu, and 5'-OH end of mt cytochrome c oxidase II) confirmed the presence of mitochondrial DNA in the secretory material in +9 hr APF prepupae ( h , i , (green)) along with F-actin ( h , j , (blue)). Although nuclear proteins are released by an apocrine mechanism into the lumen, nuclear DNA was never detected in the secretion. When in situ hybridization was performed in +9 hr APF prepupae with a probe for a nuclear gene Doa locus, signal was found only in nuclei ( k , n , (red)) together with Hoechst 33258 staining DNA ( k , l , (green)), while F-actin was detectable in the lumen ( k , m , (blue)). Remaining confocal images 400×. L in ( f ), ( g ), ( h ) and ( k )  =  lumen.

    Techniques Used: Western Blot, Isolation, In Situ, Hybridization, Sequencing, Staining

    Evidence for the graded temporal release of different proteins by apocrine secretion. ( a ) = At+8.5 hr APF, the ribosomal protein Rp40 (blue) is completely released into lumen, the cortical membrane component α-spectrin (green) was removed from the lateral and apical surfaces but remained at the basal surface, and the nuclear receptor Usp (red) is about half-released into the lumen. ( b ) At +9 hr APF, both the ribosomal protein Rp21 (green) as well as the ecdysone-inducible Ets-like E74 transcription factor (red) are present only in the lumen, whereas there remains significant F-actin (blue) signal on the cortical membranes. ( c ) At the same time (+9 hr APF), the ecdysone-regulated transcription factor and nuclear tumor suppressor are secreted differently: while Kr-h (red ( d )) is completely extruded into the lumen, p53 (green ( e )) only starts to be released and the majority of its signal is still detected in nuclei. Although filamentous actin (blue ( f )) already is being secreted into the lumen, there is detectable signal still visible on cell membranes. ( g ) During +9 to +10 hr APF, the ecdysone-regulated transcription factor BR-C (green ( h )) is completely released into the lumen, whereas lamin C (red), a component of the nuclear envelope, is only partially released and can be still detected on the nuclear membrane ( i ). Although filamentous actin (blue) is already within the lumen, significant amounts of it still line the cortical cytoskeleton, mainly at the apical membrane ( j ). ( k ) At the end of +10 hr APF both, Rab11 (green ( l )), a member of the GTPase family of membrane proteins as well as the tumor suppressor transcription factor p53 (red ( m )) have been completely secreted into the lumen. Hoechst 33258 was used to detect nuclear DNA (blue ( n )) which stays in nuclei. All confocal images 400×.
    Figure Legend Snippet: Evidence for the graded temporal release of different proteins by apocrine secretion. ( a ) = At+8.5 hr APF, the ribosomal protein Rp40 (blue) is completely released into lumen, the cortical membrane component α-spectrin (green) was removed from the lateral and apical surfaces but remained at the basal surface, and the nuclear receptor Usp (red) is about half-released into the lumen. ( b ) At +9 hr APF, both the ribosomal protein Rp21 (green) as well as the ecdysone-inducible Ets-like E74 transcription factor (red) are present only in the lumen, whereas there remains significant F-actin (blue) signal on the cortical membranes. ( c ) At the same time (+9 hr APF), the ecdysone-regulated transcription factor and nuclear tumor suppressor are secreted differently: while Kr-h (red ( d )) is completely extruded into the lumen, p53 (green ( e )) only starts to be released and the majority of its signal is still detected in nuclei. Although filamentous actin (blue ( f )) already is being secreted into the lumen, there is detectable signal still visible on cell membranes. ( g ) During +9 to +10 hr APF, the ecdysone-regulated transcription factor BR-C (green ( h )) is completely released into the lumen, whereas lamin C (red), a component of the nuclear envelope, is only partially released and can be still detected on the nuclear membrane ( i ). Although filamentous actin (blue) is already within the lumen, significant amounts of it still line the cortical cytoskeleton, mainly at the apical membrane ( j ). ( k ) At the end of +10 hr APF both, Rab11 (green ( l )), a member of the GTPase family of membrane proteins as well as the tumor suppressor transcription factor p53 (red ( m )) have been completely secreted into the lumen. Hoechst 33258 was used to detect nuclear DNA (blue ( n )) which stays in nuclei. All confocal images 400×.

    Techniques Used:

    12) Product Images from "S100A8 and S100A9 Promotes Invasion and Migration through p38 Mitogen-Activated Protein Kinase-Dependent NF-?B Activation in Gastric Cancer Cells"

    Article Title: S100A8 and S100A9 Promotes Invasion and Migration through p38 Mitogen-Activated Protein Kinase-Dependent NF-?B Activation in Gastric Cancer Cells

    Journal:

    doi: 10.1007/s10059-013-2269-x

    Effect of S100A8/A9 on gastric cancer cells. (A) Cells were treated with S100A8/A9 at the indicated concentrations for 24 h and cell death was measured by MMT assay. Data are mean ± SEM of four independent experiments. * P < 0.05 compared with control. After 24 h incubation with 1 or 30 μg/ml S100A8/A9 (A89), Apoptotic cells were evaluated by Hoechst 33258 staining (B) and cell cycle analysis (C). The percentages within box indicate the extent of sub-G 1 phase.
    Figure Legend Snippet: Effect of S100A8/A9 on gastric cancer cells. (A) Cells were treated with S100A8/A9 at the indicated concentrations for 24 h and cell death was measured by MMT assay. Data are mean ± SEM of four independent experiments. * P < 0.05 compared with control. After 24 h incubation with 1 or 30 μg/ml S100A8/A9 (A89), Apoptotic cells were evaluated by Hoechst 33258 staining (B) and cell cycle analysis (C). The percentages within box indicate the extent of sub-G 1 phase.

    Techniques Used: Incubation, Staining, Cell Cycle Assay

    Effect of S100A8/A9 on gastric cancer cell invasion and migration. (A) Cells were treated with 1 μg/ml S100A8/A9 (A89) for 24 h and cell proliferation was estimated by MTT assay. The cell invasion (B) and migration (C) were measured using a trans well assay and quantified after labeling with Hoechst 33258. Data are mean ± SEM of four independent experiments. * P <  0.05 compared with control (C).
    Figure Legend Snippet: Effect of S100A8/A9 on gastric cancer cell invasion and migration. (A) Cells were treated with 1 μg/ml S100A8/A9 (A89) for 24 h and cell proliferation was estimated by MTT assay. The cell invasion (B) and migration (C) were measured using a trans well assay and quantified after labeling with Hoechst 33258. Data are mean ± SEM of four independent experiments. * P < 0.05 compared with control (C).

    Techniques Used: Migration, MTT Assay, Labeling

    13) Product Images from "Lup-20(29)-en-3β,28-di-yl-nitrooxy acetate affects MCF-7 proliferation through the crosstalk between apoptosis and autophagy in mitochondria"

    Article Title: Lup-20(29)-en-3β,28-di-yl-nitrooxy acetate affects MCF-7 proliferation through the crosstalk between apoptosis and autophagy in mitochondria

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-017-0255-5

    NBT induced autophagic flux in MCF-7 cells. a  Representative TEM images of MCF-7 cells treated with NBT (12 μM). N nucleus; M mitochondria; red arrows indicate autophagic vacuoles (scale bars: 2 μm, 500 nm).  b  Total cellular extract of MCF-7 cells treated with 6, 12, and 24 μM NBT was prepared and subjected to Western blot by using antibodies against Atg5, LC3B, and p62. Data represent mean ± SD of three independent experiments.  c  Cells were transfected with a tandem reporter construct (tfLC3) for 48 h and were exposed to Rapa (0.25 μM), NBT (12 μM), and/or CQ (40 μM) for 24 h. The cells were then stained with Hoechst 33258 fluorescent dye. The puncta were examined by using a fluorescence microscope. Scale bars: 50 μm
    Figure Legend Snippet: NBT induced autophagic flux in MCF-7 cells. a Representative TEM images of MCF-7 cells treated with NBT (12 μM). N nucleus; M mitochondria; red arrows indicate autophagic vacuoles (scale bars: 2 μm, 500 nm). b Total cellular extract of MCF-7 cells treated with 6, 12, and 24 μM NBT was prepared and subjected to Western blot by using antibodies against Atg5, LC3B, and p62. Data represent mean ± SD of three independent experiments. c Cells were transfected with a tandem reporter construct (tfLC3) for 48 h and were exposed to Rapa (0.25 μM), NBT (12 μM), and/or CQ (40 μM) for 24 h. The cells were then stained with Hoechst 33258 fluorescent dye. The puncta were examined by using a fluorescence microscope. Scale bars: 50 μm

    Techniques Used: Transmission Electron Microscopy, Western Blot, Transfection, Construct, Staining, Fluorescence, Microscopy

    NBT induced MCF-7 cell apoptosis through the mitochondrial pathway. a  MCF-7 cells were treated with various concentrations of NBT for 24 h, and apoptosis was examined by using annexin V with PI staining with flow cytometry.  b  Hoechst 33258 staining assay showed that NBT at doses of 6, 12, and 24 μM induced chromatin shrinking of MCF-7 cells (×200, scale bars: 100 μm; ×400, scale bars: 50  μm).  c  Treatment of 6, 12, and 24 μM NBT influenced caspase-9 activity in MCF-7 cells.  d  Total cellular extract of MCF-7 cells treated with 6, 12, and 24 μM NBT was prepared and subjected to Western blot by using an antibody against PARP.  e  JC-1 staining assay determined the ΔΨm in mitochondria of MCF-7 cells treated with 6, 12, and 24 μM NBT.  f  Total cellular extract, mitochondrial fraction, and cytosol fractions of MCF-7 cells treated with 6, 12, and 24 μM NBT were prepared and subjected to Western blot by using antibodies against Bcl-2, Bax, and Cyt C (mitochondrial and cytosol fraction).  g  Total cellular extract of MCF-7 cells treated with 6, 12, and 24 μM NBT was prepared and subjected to Western blot by using antibodies against DRP1, Fis1, and Mfn2. Data represent mean ± SD of three independent experiments ( * P 
    Figure Legend Snippet: NBT induced MCF-7 cell apoptosis through the mitochondrial pathway. a MCF-7 cells were treated with various concentrations of NBT for 24 h, and apoptosis was examined by using annexin V with PI staining with flow cytometry. b Hoechst 33258 staining assay showed that NBT at doses of 6, 12, and 24 μM induced chromatin shrinking of MCF-7 cells (×200, scale bars: 100 μm; ×400, scale bars: 50  μm). c Treatment of 6, 12, and 24 μM NBT influenced caspase-9 activity in MCF-7 cells. d Total cellular extract of MCF-7 cells treated with 6, 12, and 24 μM NBT was prepared and subjected to Western blot by using an antibody against PARP. e JC-1 staining assay determined the ΔΨm in mitochondria of MCF-7 cells treated with 6, 12, and 24 μM NBT. f Total cellular extract, mitochondrial fraction, and cytosol fractions of MCF-7 cells treated with 6, 12, and 24 μM NBT were prepared and subjected to Western blot by using antibodies against Bcl-2, Bax, and Cyt C (mitochondrial and cytosol fraction). g Total cellular extract of MCF-7 cells treated with 6, 12, and 24 μM NBT was prepared and subjected to Western blot by using antibodies against DRP1, Fis1, and Mfn2. Data represent mean ± SD of three independent experiments ( * P 

    Techniques Used: Staining, Flow Cytometry, Cytometry, Activity Assay, Western Blot

    14) Product Images from "Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons"

    Article Title: Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons

    Journal: Neural Regeneration Research

    doi: 10.3969/j.issn.1673-5374.2013.02.003

    Apoptotic neurons in rat hippocampus. (A) Morphology of cultured rat hippocampal neurons following DNA fluorescent staining (Hoechst 33258 staining, inverted fluorescence microscope, × 200). Arrows indicate apoptotic neurons (Hoechst 33258- positive cells displayed strong blue fluorescence). Apoptotic cells were evident in the 3-morpholinosydnonimine (SIN-1; 1.0 mM) group, but fewer were visible in the SIN-1 + DIDS group. (B) Quantification of apoptotic cells.  a P
    Figure Legend Snippet: Apoptotic neurons in rat hippocampus. (A) Morphology of cultured rat hippocampal neurons following DNA fluorescent staining (Hoechst 33258 staining, inverted fluorescence microscope, × 200). Arrows indicate apoptotic neurons (Hoechst 33258- positive cells displayed strong blue fluorescence). Apoptotic cells were evident in the 3-morpholinosydnonimine (SIN-1; 1.0 mM) group, but fewer were visible in the SIN-1 + DIDS group. (B) Quantification of apoptotic cells. a P

    Techniques Used: Cell Culture, Staining, Fluorescence, Microscopy

    Morphology of rat hippocampal neurons 12 days after culture (inverted fluorescence microscope, × 200). (A) Hoechst 33258 staining shows that normal neurons stained blue (Hoechst 33258 in blue). (B) The neuronal antibody anti-NeuN was indicated by red staining (Cy3 labeling in red). (C) Merged image of A and B. Neurons were simultaneously positive for Hoechst 33258 and the neuron-specific antibody NeuN.
    Figure Legend Snippet: Morphology of rat hippocampal neurons 12 days after culture (inverted fluorescence microscope, × 200). (A) Hoechst 33258 staining shows that normal neurons stained blue (Hoechst 33258 in blue). (B) The neuronal antibody anti-NeuN was indicated by red staining (Cy3 labeling in red). (C) Merged image of A and B. Neurons were simultaneously positive for Hoechst 33258 and the neuron-specific antibody NeuN.

    Techniques Used: Fluorescence, Microscopy, Staining, Labeling

    15) Product Images from "The effect of hypoxia-inducible factor 1-alpha on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes"

    Article Title: The effect of hypoxia-inducible factor 1-alpha on hypoxia-induced apoptosis in primary neonatal rat ventricular myocytes

    Journal: Cardiovascular Journal of Africa

    doi:

    Hypoxia strongly induced apoptosis in primary neonatal rat ventricular myocytes in a degree-dependent manner. Apoptosis in cells cultured for 24 hours under normoxic (20% O 2 ) conditions and different degrees of hypoxia (5% O 2 , 2% O 2  and 1% O 2 ). (A) Hoechst 33258 staining results. Nuclei of apoptotic cells appeared to be intensely fluorescent, fragmented and condensed. (B) Hoechst 33258-positive apoptotic cells were quantified in terms of cells per high-power field. Original magnification was × 400. Hypoxia treatment significantly increased the number of apoptotic cells (# p
    Figure Legend Snippet: Hypoxia strongly induced apoptosis in primary neonatal rat ventricular myocytes in a degree-dependent manner. Apoptosis in cells cultured for 24 hours under normoxic (20% O 2 ) conditions and different degrees of hypoxia (5% O 2 , 2% O 2 and 1% O 2 ). (A) Hoechst 33258 staining results. Nuclei of apoptotic cells appeared to be intensely fluorescent, fragmented and condensed. (B) Hoechst 33258-positive apoptotic cells were quantified in terms of cells per high-power field. Original magnification was × 400. Hypoxia treatment significantly increased the number of apoptotic cells (# p

    Techniques Used: Cell Culture, Staining

    Hypoxia-induced apoptosis decreased after inhibition of HIF-1α in primary neonatal rat ventricular myocytes. Cells were subjected to normoxia (20% O 2 ), hypoxic (1% O 2 ) and hypoxic (1% O 2 ) condition in the presence of YC-1 for 24 hours. (A) Hoechst 33258 staining of cultures under (1) normoxia, (2) hypoxia, and (3) hypoxia in the presence of YC-1. (B) Hoechst 33258-positive apoptotic cells were quantified (per high-power field). Original magnification was × 400. YC-1 significantly decreased the apoptosis induced by hypoxia (# p
    Figure Legend Snippet: Hypoxia-induced apoptosis decreased after inhibition of HIF-1α in primary neonatal rat ventricular myocytes. Cells were subjected to normoxia (20% O 2 ), hypoxic (1% O 2 ) and hypoxic (1% O 2 ) condition in the presence of YC-1 for 24 hours. (A) Hoechst 33258 staining of cultures under (1) normoxia, (2) hypoxia, and (3) hypoxia in the presence of YC-1. (B) Hoechst 33258-positive apoptotic cells were quantified (per high-power field). Original magnification was × 400. YC-1 significantly decreased the apoptosis induced by hypoxia (# p

    Techniques Used: Inhibition, Staining

    16) Product Images from "DNA orientation-specific adhesion and patterning of living mammalian cells on self-assembled DNA monolayers †Electronic supplementary information (ESI) available: Details in experimental section and supporting figures. See DOI: 10.1039/c5sc04102cClick here for additional data file."

    Article Title: DNA orientation-specific adhesion and patterning of living mammalian cells on self-assembled DNA monolayers †Electronic supplementary information (ESI) available: Details in experimental section and supporting figures. See DOI: 10.1039/c5sc04102cClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c5sc04102c

    Patterns of living cells on DNA-SAM substrate. SH-T20 was spotted on gold substrate by a microarrayer to form a spot matrix (500 μm distance) or four English letters: “CELL”; after passivation with SH-OEG; MCF-7 cells were seeded and cultured overnight. After staining with Hoechst 33258 and phalloidin–TRITC, cell patterns were observed by microscope. (a) English letters “CELL” built by patterned cells. (b) Cell spot matrix. Scale bars: 200 μm.
    Figure Legend Snippet: Patterns of living cells on DNA-SAM substrate. SH-T20 was spotted on gold substrate by a microarrayer to form a spot matrix (500 μm distance) or four English letters: “CELL”; after passivation with SH-OEG; MCF-7 cells were seeded and cultured overnight. After staining with Hoechst 33258 and phalloidin–TRITC, cell patterns were observed by microscope. (a) English letters “CELL” built by patterned cells. (b) Cell spot matrix. Scale bars: 200 μm.

    Techniques Used: Cell Culture, Staining, Microscopy

    The (a) scheme and (b) fluorescence microscopy images of control cell adhesion through ATP. MCF-7 cell was seeded on DNA SAMs either in the presence or absence of ATP overnight. Cells were fixed and nuclei (blue) and actin (green) were labelled with Hoechst 33258 and phalloidin–TRITC. Scale bars: 200 μm. We constructed this ATP-responsive DNA SAM by grafting ATP's aptamer on gold substrate. Without ATP, the aptamer adopted unfolded state and tended to stay upright due to strong lateral electrostatic repulsion, which has high cell adhesion property. With ATP, the conformational change of aptamer forced it to adopt a folded structure, which was unfavorable for cell adhesion.
    Figure Legend Snippet: The (a) scheme and (b) fluorescence microscopy images of control cell adhesion through ATP. MCF-7 cell was seeded on DNA SAMs either in the presence or absence of ATP overnight. Cells were fixed and nuclei (blue) and actin (green) were labelled with Hoechst 33258 and phalloidin–TRITC. Scale bars: 200 μm. We constructed this ATP-responsive DNA SAM by grafting ATP's aptamer on gold substrate. Without ATP, the aptamer adopted unfolded state and tended to stay upright due to strong lateral electrostatic repulsion, which has high cell adhesion property. With ATP, the conformational change of aptamer forced it to adopt a folded structure, which was unfavorable for cell adhesion.

    Techniques Used: Fluorescence, Microscopy, Construct

    The (a) scheme, (b) fluorescence microscopy images and (c) statistics of adhered cell numbers of MCF-7 cells on gold surface grafting with thiol-oligoT of different lengths. DNA SAMs were formed by thiol-oligoT with different lengths (T20, T10, T5, T2) followed by passivation with SH-OEG, MCF-7 cell was cultured on those SAMs overnight and stained with Hoechst 33258 and phalloidin–TRITC. Cell number was analyzed by ImageJ. Compared to T20 SAMs, no significant difference on cell density is observed for T10 SAMs. However, cell number decreased significantly for T5 SAMs and almost no cells attached for T2 SAMs. Cells in 0.6 mm 2  were counted. Scale bars: 200 μm.
    Figure Legend Snippet: The (a) scheme, (b) fluorescence microscopy images and (c) statistics of adhered cell numbers of MCF-7 cells on gold surface grafting with thiol-oligoT of different lengths. DNA SAMs were formed by thiol-oligoT with different lengths (T20, T10, T5, T2) followed by passivation with SH-OEG, MCF-7 cell was cultured on those SAMs overnight and stained with Hoechst 33258 and phalloidin–TRITC. Cell number was analyzed by ImageJ. Compared to T20 SAMs, no significant difference on cell density is observed for T10 SAMs. However, cell number decreased significantly for T5 SAMs and almost no cells attached for T2 SAMs. Cells in 0.6 mm 2 were counted. Scale bars: 200 μm.

    Techniques Used: Fluorescence, Microscopy, Cell Culture, Staining

    (a) The scheme of substrates derived from different DNA bases and showing different preferences to cell adhesion. (b) Fluorescence microscopy images of MCF-7 cell adhesion on different substrates. MCF-7 cell was seeded on different substrates and cultured overnight, then the cell was fixed and labeled for nuclei (blue) and actin (green) by Hoechst 33258 and phalloidin–TRITC, respectively. All the gold substrates were passivated with SH-OEG after grafting with different thiolated 20-mer DNA strands. On SH-OEG and SH-A20 substrates, no cell was observed. However, on DNA SAMs substrates formed by other thiolated DNA strands (T20, C20, G20 and a random sequence), the number of attached cells is considerable. Scale bars: 200 μm. Cells in 0.6 mm 2  were counted. (c) The statistics of adhered cell numbers on these substrates.
    Figure Legend Snippet: (a) The scheme of substrates derived from different DNA bases and showing different preferences to cell adhesion. (b) Fluorescence microscopy images of MCF-7 cell adhesion on different substrates. MCF-7 cell was seeded on different substrates and cultured overnight, then the cell was fixed and labeled for nuclei (blue) and actin (green) by Hoechst 33258 and phalloidin–TRITC, respectively. All the gold substrates were passivated with SH-OEG after grafting with different thiolated 20-mer DNA strands. On SH-OEG and SH-A20 substrates, no cell was observed. However, on DNA SAMs substrates formed by other thiolated DNA strands (T20, C20, G20 and a random sequence), the number of attached cells is considerable. Scale bars: 200 μm. Cells in 0.6 mm 2 were counted. (c) The statistics of adhered cell numbers on these substrates.

    Techniques Used: Derivative Assay, Fluorescence, Microscopy, Cell Culture, Labeling, Sequencing

    17) Product Images from "Adenosine A1 receptor: A neuroprotective target in light induced retinal degeneration"

    Article Title: Adenosine A1 receptor: A neuroprotective target in light induced retinal degeneration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198838

    Double immunolabeling for Iba 1 and MHC-II. Representative sections of CPA Control retina (top row); CPA treated retina (second row); DPCPX Control retina (third row) and DPCPX treated retina (fourth row). In every case nuclear staining with Hoechst 33258 (blue), Iba 1 immunolabeling (green), MHC II immunostaining (red) and double labeling (merge) of the same sections may be observed form left to right. Insets show higher magnifications of merge images. Arrow heads show reactive microglial cells. Observe the low number of double stained reactive microglial cells in CPA treated retina and the higher number of double stained reactive microglial cells in DPCPX treated retina. Scale bars = 20 μm and 5μm (inset).
    Figure Legend Snippet: Double immunolabeling for Iba 1 and MHC-II. Representative sections of CPA Control retina (top row); CPA treated retina (second row); DPCPX Control retina (third row) and DPCPX treated retina (fourth row). In every case nuclear staining with Hoechst 33258 (blue), Iba 1 immunolabeling (green), MHC II immunostaining (red) and double labeling (merge) of the same sections may be observed form left to right. Insets show higher magnifications of merge images. Arrow heads show reactive microglial cells. Observe the low number of double stained reactive microglial cells in CPA treated retina and the higher number of double stained reactive microglial cells in DPCPX treated retina. Scale bars = 20 μm and 5μm (inset).

    Techniques Used: Immunolabeling, Staining, Immunostaining, Labeling

    Double immunolabeling for Iba 1 and A1 receptor. Representative sections of CPA Control retina (top row); CPA treated retina (second row); DPCPX Control retina (third row) and DPCPX treated retina (fourth row). In every case nuclear staining with Hoechst 33258 (blue), A1 receptor immunolabeling (green); Iba 1 immunolabeling (red), and double labeling of the same sections may be observed form left to right. Insets show higher magnifications of merge images. Scale bars = 20 μm.
    Figure Legend Snippet: Double immunolabeling for Iba 1 and A1 receptor. Representative sections of CPA Control retina (top row); CPA treated retina (second row); DPCPX Control retina (third row) and DPCPX treated retina (fourth row). In every case nuclear staining with Hoechst 33258 (blue), A1 receptor immunolabeling (green); Iba 1 immunolabeling (red), and double labeling of the same sections may be observed form left to right. Insets show higher magnifications of merge images. Scale bars = 20 μm.

    Techniques Used: Immunolabeling, Staining, Labeling

    18) Product Images from "Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2"

    Article Title: Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00186

    Tis21-retrovirus inhibits the deregulated proliferation of dentate gyrus progenitor cells and rescues the defective terminal differentiation of Tis21-null neurons.  (A)  Scheme of retrovirus infection timeline, structure, and injection area.  (B)  Representative confocal images (40× magnification) of coronal sections of the dentate gyrus, labeled with Hoechst 33258, Ki67 and with GFP, 5 days after infection with either GFP-Tis21 or GFP-empty retroviruses. Scale bars, 50 μm. The white arrowheads indicate cells positive for both GFP and Ki67, white arrows cells positive only for GFP.  (C)  Percentage ratio between GFP + Ki67 +  cells and the total number of infected cells (GFP + ), from the analysis of Tis21 knockout dentate gyrus infected with either GFP-Tis21 or GFP-empty virus. The percentage of dividing cells (Ki67 + ) is reduced by the Tis21 virus, relative to the empty virus infections. * p
    Figure Legend Snippet: Tis21-retrovirus inhibits the deregulated proliferation of dentate gyrus progenitor cells and rescues the defective terminal differentiation of Tis21-null neurons. (A) Scheme of retrovirus infection timeline, structure, and injection area. (B) Representative confocal images (40× magnification) of coronal sections of the dentate gyrus, labeled with Hoechst 33258, Ki67 and with GFP, 5 days after infection with either GFP-Tis21 or GFP-empty retroviruses. Scale bars, 50 μm. The white arrowheads indicate cells positive for both GFP and Ki67, white arrows cells positive only for GFP. (C) Percentage ratio between GFP + Ki67 + cells and the total number of infected cells (GFP + ), from the analysis of Tis21 knockout dentate gyrus infected with either GFP-Tis21 or GFP-empty virus. The percentage of dividing cells (Ki67 + ) is reduced by the Tis21 virus, relative to the empty virus infections. * p

    Techniques Used: Infection, Injection, Labeling, Knock-Out

    NeuroD2-retrovirus rescues the defective terminal differentiation of Tis21-null neurons without affecting the proliferation of progenitor cells.  (A)  Retrovirus infection timeline, structure and injection area.  (B)  Representative confocal images (40× magnification) of coronal sections of the dentate gyrus, labeled with Hoechst 33258, Ki67 and with GFP, 5 days after infection with either GFP-NeuroD2 or GFP-empty retroviruses. Scale bars, 50 μm. The white arrowheads indicate cells positive for both GFP and Ki67, white arrows cells positive only for GFP.  (C)  Percentage ratio between GFP + Ki67 +  cells and the total number of infected cells (GFP + ), in Tis21 knockout dentate gyrus infected with GFP-NeuroD2 or GFP-empty retroviruses. The percentage of dividing cells (Ki67 + ) is reduced, although not significantly, by the NeuroD2 virus, relative to control virus infections. NS,  p >  0.05 vs. cells infected with GFP-empty retrovirus; Mann-Withney U test.  (D)  Representative confocal images of dentate gyrus cells triple-labeled with Calretinin, NeuN and GFP, 5 days after infection with either GFP-NeuroD2 or GFP-empty retroviruses. Scale bars, 50 μm. White arrows: infected terminally differentiated neurons (GFP + Calretinin − NeuN + ); arrowheads: infected stage 5 immature neurons (GFP + Calretinin + NeuN + ).  (E)  Percentage ratio of stage 5 immature neurons (GFP + stage 5) or stage 6 terminally differentiated neurons (GFP + stage 6) to the total number of infected cells (GFP + ), analyzed in Tis21 knockout dentate gyrus infected with either GFP-NeuroD2 or GFP-empty retrovirus. The higher percentage of stage 5 and the lower percentage of stage 6 control-infected neurons are equalized by the NeuroD2 virus. * p
    Figure Legend Snippet: NeuroD2-retrovirus rescues the defective terminal differentiation of Tis21-null neurons without affecting the proliferation of progenitor cells. (A) Retrovirus infection timeline, structure and injection area. (B) Representative confocal images (40× magnification) of coronal sections of the dentate gyrus, labeled with Hoechst 33258, Ki67 and with GFP, 5 days after infection with either GFP-NeuroD2 or GFP-empty retroviruses. Scale bars, 50 μm. The white arrowheads indicate cells positive for both GFP and Ki67, white arrows cells positive only for GFP. (C) Percentage ratio between GFP + Ki67 + cells and the total number of infected cells (GFP + ), in Tis21 knockout dentate gyrus infected with GFP-NeuroD2 or GFP-empty retroviruses. The percentage of dividing cells (Ki67 + ) is reduced, although not significantly, by the NeuroD2 virus, relative to control virus infections. NS, p > 0.05 vs. cells infected with GFP-empty retrovirus; Mann-Withney U test. (D) Representative confocal images of dentate gyrus cells triple-labeled with Calretinin, NeuN and GFP, 5 days after infection with either GFP-NeuroD2 or GFP-empty retroviruses. Scale bars, 50 μm. White arrows: infected terminally differentiated neurons (GFP + Calretinin − NeuN + ); arrowheads: infected stage 5 immature neurons (GFP + Calretinin + NeuN + ). (E) Percentage ratio of stage 5 immature neurons (GFP + stage 5) or stage 6 terminally differentiated neurons (GFP + stage 6) to the total number of infected cells (GFP + ), analyzed in Tis21 knockout dentate gyrus infected with either GFP-NeuroD2 or GFP-empty retrovirus. The higher percentage of stage 5 and the lower percentage of stage 6 control-infected neurons are equalized by the NeuroD2 virus. * p

    Techniques Used: Infection, Injection, Labeling, Knock-Out

    shId3-retrovirus rescues the defective terminal differentiation of Tis21-null neurons without effects on proliferating cells.  (A)  Retrovirus infection timeline, structure and injection area.  (B)  Representative confocal images (40× magnification) of coronal sections of the dentate gyrus, labeled with Hoechst 33258 and with Ki67 and GFP, 5 days after infection with either GFP-shId3 or GFP-shLUC (control) retroviruses. Scale bars, 50 μm. The white arrowheads indicate cells positive for both GFP and Ki67.  (C)  Percentage ratio between GFP + Ki67 +  cells and the total number of infected cells (GFP + ), in Tis21 knockout dentate gyrus infected with GFP-shId3 or GFP-shLUC virus. The ratio of dividing cells (Ki67 + ) is not changed by the shId3 virus, relative to the shLUC control virus infections. NS,  p >  0.05 vs. cells infected with GFP-shLUCvirus; Mann-Withney U test.  (D)  Representative confocal images of dentate gyrus cells triple-labeled with Calretinin, NeuN and GFP, 5 days after infection with either GFP-shId3 or GFP-shLUC retroviruses. Scale bars, 50 μm. White arrows indicate infected terminally differentiated neurons (GFP + Calretinin − NeuN + ); arrowheads indicate infected stage 5 immature neurons (GFP + Calretinin + NeuN + ).  (E)  Percentage ratio between stage 5 immature neurons (GFP + stage 5) or stage 6 terminally differentiated neurons (GFP + stage 6) and the total number of infected cells (GFP + ), analyzed in Tis21 knockout dentate gyrus infected with either GFP-shId3 or GFP-shLUC virus. The percentages of stage 5 neurons or stage 6 neurons observed in the shLUC virus infections are significantly decreased or increased, respectively, by the shId3 virus. * p
    Figure Legend Snippet: shId3-retrovirus rescues the defective terminal differentiation of Tis21-null neurons without effects on proliferating cells. (A) Retrovirus infection timeline, structure and injection area. (B) Representative confocal images (40× magnification) of coronal sections of the dentate gyrus, labeled with Hoechst 33258 and with Ki67 and GFP, 5 days after infection with either GFP-shId3 or GFP-shLUC (control) retroviruses. Scale bars, 50 μm. The white arrowheads indicate cells positive for both GFP and Ki67. (C) Percentage ratio between GFP + Ki67 + cells and the total number of infected cells (GFP + ), in Tis21 knockout dentate gyrus infected with GFP-shId3 or GFP-shLUC virus. The ratio of dividing cells (Ki67 + ) is not changed by the shId3 virus, relative to the shLUC control virus infections. NS, p > 0.05 vs. cells infected with GFP-shLUCvirus; Mann-Withney U test. (D) Representative confocal images of dentate gyrus cells triple-labeled with Calretinin, NeuN and GFP, 5 days after infection with either GFP-shId3 or GFP-shLUC retroviruses. Scale bars, 50 μm. White arrows indicate infected terminally differentiated neurons (GFP + Calretinin − NeuN + ); arrowheads indicate infected stage 5 immature neurons (GFP + Calretinin + NeuN + ). (E) Percentage ratio between stage 5 immature neurons (GFP + stage 5) or stage 6 terminally differentiated neurons (GFP + stage 6) and the total number of infected cells (GFP + ), analyzed in Tis21 knockout dentate gyrus infected with either GFP-shId3 or GFP-shLUC virus. The percentages of stage 5 neurons or stage 6 neurons observed in the shLUC virus infections are significantly decreased or increased, respectively, by the shId3 virus. * p

    Techniques Used: Infection, Injection, Labeling, Knock-Out

    19) Product Images from "Single-step assembly of polymer-lipid hybrid nanoparticles for mitomycin C delivery"

    Article Title: Single-step assembly of polymer-lipid hybrid nanoparticles for mitomycin C delivery

    Journal: Nanoscale Research Letters

    doi: 10.1186/1556-276X-9-560

    Intracellular drug delivery of the hybrid PLA NPs/MMC-SPC.  Confocal laser scanning microscopy images of H 22  cells incubated with the  (A,B,C)  coumarin-6-PLA NPs/FITC-MMC-SPC and  (D,E,F)  coumarin-6-PLA NPs/FITC-MMC at the equivalent coumarin-6 concentration for 12 h at 37°C.  (A,D)  Left column showed the red fluorescence from the cytoskeleton (the cell nuclei were stained with rhodamine-phalloidin).  (B,E)  Middle column showed the green fluorescence from the drugs (the drugs were labeled with FITC). The merged image of the images in left column and middle column is shown in  (E,F)  right column. The nuclei were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) (blue).
    Figure Legend Snippet: Intracellular drug delivery of the hybrid PLA NPs/MMC-SPC. Confocal laser scanning microscopy images of H 22 cells incubated with the (A,B,C) coumarin-6-PLA NPs/FITC-MMC-SPC and (D,E,F) coumarin-6-PLA NPs/FITC-MMC at the equivalent coumarin-6 concentration for 12 h at 37°C. (A,D) Left column showed the red fluorescence from the cytoskeleton (the cell nuclei were stained with rhodamine-phalloidin). (B,E) Middle column showed the green fluorescence from the drugs (the drugs were labeled with FITC). The merged image of the images in left column and middle column is shown in (E,F) right column. The nuclei were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) (blue).

    Techniques Used: Proximity Ligation Assay, Confocal Laser Scanning Microscopy, Incubation, Concentration Assay, Fluorescence, Staining, Labeling

    In vitro  cellular uptake of the hybrid PLA NPs/MMC-SPC. (A)  Confocal laser scanning microscopy images of H 22  cells incubated with the  (a, b, and c)  coumarin-6-PLA NPs/MMC-SPC and  (d, e, and f)  coumarin-6-PLA NPs/MMC at the equivalent coumarin-6 concentration for 6 h at 37°C.  (a and d)  Left column showed the blue fluorescence from the cell nuclei (the cell nuclei were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA)).  (b and e)  Middle column showed the green fluorescence from the NPs (the NPs were labeled with coumarin-6).  (c and f)  Right column is the merged picture of the left column and right column. All the scale bars represented 25 μm. All images were taken under identical instrumental conditions.  (B)  Flow cytometer tests of H 22  cells incubated with the coumarin-6-PLA NPs/MMC-SPC and coumarin-6-PLA NPs/MMC (equivalent coumarin-6 concentration) for 0.5, 1.5, and 4.5 h. Data are presented as mean ± SD ( n  =3). * P
    Figure Legend Snippet: In vitro cellular uptake of the hybrid PLA NPs/MMC-SPC. (A) Confocal laser scanning microscopy images of H 22 cells incubated with the (a, b, and c) coumarin-6-PLA NPs/MMC-SPC and (d, e, and f) coumarin-6-PLA NPs/MMC at the equivalent coumarin-6 concentration for 6 h at 37°C. (a and d) Left column showed the blue fluorescence from the cell nuclei (the cell nuclei were stained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA)). (b and e) Middle column showed the green fluorescence from the NPs (the NPs were labeled with coumarin-6). (c and f) Right column is the merged picture of the left column and right column. All the scale bars represented 25 μm. All images were taken under identical instrumental conditions. (B) Flow cytometer tests of H 22 cells incubated with the coumarin-6-PLA NPs/MMC-SPC and coumarin-6-PLA NPs/MMC (equivalent coumarin-6 concentration) for 0.5, 1.5, and 4.5 h. Data are presented as mean ± SD ( n =3). * P

    Techniques Used: In Vitro, Proximity Ligation Assay, Confocal Laser Scanning Microscopy, Incubation, Concentration Assay, Fluorescence, Staining, Labeling, Flow Cytometry, Cytometry

    20) Product Images from "NAPO as a novel marker for apoptosis"

    Article Title: NAPO as a novel marker for apoptosis

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200106044

    NAPO  antigen is positive in senescent cells.  Presenescent (A, C, and E) and senescent (B, D, and F) MRC-5 cells were stained for senescence-associated β-galactosidase activity (A and B),  NAPO  immunoreactivity (C and D), and Hoechst 33258 DNA staining (E and F). Note that senescence-associated β-galactosidase-positive cells are also positive for  NAPO  antigen.
    Figure Legend Snippet: NAPO antigen is positive in senescent cells. Presenescent (A, C, and E) and senescent (B, D, and F) MRC-5 cells were stained for senescence-associated β-galactosidase activity (A and B), NAPO immunoreactivity (C and D), and Hoechst 33258 DNA staining (E and F). Note that senescence-associated β-galactosidase-positive cells are also positive for NAPO antigen.

    Techniques Used: Staining, Activity Assay

    NAPO  expression during cell cycle.  Huh7 cells were synchronized by nocodazole treatment, followed by mitotic shake-off. Freshly collected cells were then grown in culture for up to 36 h. The S phase was identified by BrdU incorporation assay. Time points between 0–16, 20–32, and 36 h were evaluated respectively as G1, S, and G2 phases according to the BrdU incorporation index (A).  NAPO  and BrdU staining were performed at 4 h intervals. B, D, F, and H illustrate  NAPO  staining of cells at mitotic arrest (B), 8 h (D), 24 h (F), and 36 h (H), respectively. C, E, G, and I show Hoechst 33258 counterstaining. Note diffused  NAPO  staining in mitotically arrested Huh7 cells (B and C) which were digitally magnified threefold for better visualization.
    Figure Legend Snippet: NAPO expression during cell cycle. Huh7 cells were synchronized by nocodazole treatment, followed by mitotic shake-off. Freshly collected cells were then grown in culture for up to 36 h. The S phase was identified by BrdU incorporation assay. Time points between 0–16, 20–32, and 36 h were evaluated respectively as G1, S, and G2 phases according to the BrdU incorporation index (A). NAPO and BrdU staining were performed at 4 h intervals. B, D, F, and H illustrate NAPO staining of cells at mitotic arrest (B), 8 h (D), 24 h (F), and 36 h (H), respectively. C, E, G, and I show Hoechst 33258 counterstaining. Note diffused NAPO staining in mitotically arrested Huh7 cells (B and C) which were digitally magnified threefold for better visualization.

    Techniques Used: Expressing, BrdU Incorporation Assay, BrdU Staining, Staining

    NAPO  antigen is positive in quiescent cells.  MRC-5 cells were tested in parallel for BrdU incorporation (A and E) or  NAPO  antigen (C and G). Cells in A–D were grown under standard culture conditions. Cells in panels E–H were serum starved for 3 d to induce a quiescent state, as indicated by negative BrdU staining in E. Note that both actively growing (C) and quiescent cells (G) are positive for  NAPO . B, D, F, and H show Hoechst 33258 counterstaining.
    Figure Legend Snippet: NAPO antigen is positive in quiescent cells. MRC-5 cells were tested in parallel for BrdU incorporation (A and E) or NAPO antigen (C and G). Cells in A–D were grown under standard culture conditions. Cells in panels E–H were serum starved for 3 d to induce a quiescent state, as indicated by negative BrdU staining in E. Note that both actively growing (C) and quiescent cells (G) are positive for NAPO . B, D, F, and H show Hoechst 33258 counterstaining.

    Techniques Used: BrdU Incorporation Assay, BrdU Staining

    Initial characterization of  NAPO  antigen.  Anti- NAPO  monoclonal antibody recognizes two bands migrating at ∼60 and 70 kD. [ 35 S]methionine-labeled Huh7 cells were subjected to immunoprecipitation with anti- NAPO  antibody (+). (−) is a negative control (A). Immunofluorescence staining of Huh7 cells with anti- NAPO  antibody indicates that  NAPO  is a nuclear antigen (B). Hoechst 33258 counterstain for nuclear DNA (C).  NAPO  immunofluorescence staining of SNU 398 cells growing in standard culture medium indicates that the majority of cell nuclei are positive, but occasionally some cells with small nuclei (presumably apoptotic) are negative (D), as indicated by white arrows in nuclear DNA staining (E).  NAPO  antigen is negative in apoptotic SNU 398 cells which are induced by growth in serum-free medium (F). Apoptotic cells are indicated by white arrows in Hoechst 33258 counterstaining (G).
    Figure Legend Snippet: Initial characterization of NAPO antigen. Anti- NAPO monoclonal antibody recognizes two bands migrating at ∼60 and 70 kD. [ 35 S]methionine-labeled Huh7 cells were subjected to immunoprecipitation with anti- NAPO antibody (+). (−) is a negative control (A). Immunofluorescence staining of Huh7 cells with anti- NAPO antibody indicates that NAPO is a nuclear antigen (B). Hoechst 33258 counterstain for nuclear DNA (C). NAPO immunofluorescence staining of SNU 398 cells growing in standard culture medium indicates that the majority of cell nuclei are positive, but occasionally some cells with small nuclei (presumably apoptotic) are negative (D), as indicated by white arrows in nuclear DNA staining (E). NAPO antigen is negative in apoptotic SNU 398 cells which are induced by growth in serum-free medium (F). Apoptotic cells are indicated by white arrows in Hoechst 33258 counterstaining (G).

    Techniques Used: Labeling, Immunoprecipitation, Negative Control, Immunofluorescence, Staining

    Identification of  NAPO  as a common apoptosis marker. NAPO  is negative in 100 μM H 2 O 2 -treated apoptotic Huh7 cells (A), in contrast to positive staining with TUNEL (C). NAPO is also lost in Fas-mediated apoptosis in Jurkat cells (E), H 2 O 2 -mediated apoptosis in 293 cells (G), and UV-C–mediated apoptosis in MRC-5 cells (I). B, D, F, H, and J show Hoechst 33258 counterstaining.
    Figure Legend Snippet: Identification of NAPO as a common apoptosis marker. NAPO is negative in 100 μM H 2 O 2 -treated apoptotic Huh7 cells (A), in contrast to positive staining with TUNEL (C). NAPO is also lost in Fas-mediated apoptosis in Jurkat cells (E), H 2 O 2 -mediated apoptosis in 293 cells (G), and UV-C–mediated apoptosis in MRC-5 cells (I). B, D, F, H, and J show Hoechst 33258 counterstaining.

    Techniques Used: Marker, Staining, TUNEL Assay

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    Article Snippet: Paragraph title: Immunofluorescence staining ... Cells were stained with rabbit anti-Bcl-2 antibody (Abcam) or mouse anti-Stat1 (phospho Y701) antibody and then with DyLightTM 488 donkey anti-rabbit IgG (Biolegend) and DyLightTM 488 goat anti-mouse IgG (Biolegend), respectively, followed by 5μg/ml of Hoechst 33258 (Sigma) staining.

    Article Title: Contribution of Schwann Cells to Remyelination in a Naturally Occurring Canine Model of CNS Neuroinflammation
    Article Snippet: On representative sections, immunofluorescence double staining procedures were performed as described [ ] on 3 μm thick paraffin-embedded sections to demonstrate a possible co-localization of p75NTR with PDGFR-α, the transcription factors Sox2 and Egr2/Krox20, GAP-43, GFAP, and periaxin. .. Nuclear counterstaining was performed with 0.01% bisbenzimide (H33258, Sigma Aldrich, Taufkirchen, Germany) and sections were mounted with Dako Fluorescent Mounting medium (DakoCytomation, Hamburg, Germany).

    Article Title: Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis
    Article Snippet: Paragraph title: Immunofluorescence ... In the last wash, 1 μg/ml of Hoechst 33258 (Sigma) was added to the cells for 10 min at room temperature.

    Article Title: Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques
    Article Snippet: Paragraph title: Indirect immunofluorescence ... All the incubations were performed at room temperature for 1 h. Cells were then counterstained for DNA with 0.1 µg/mL of Hoechst 33258 (Sigma-Aldrich, Milano, Italy) for 10 min, washed with PBS, and mounted in a drop of Mowiol (Calbiochem-Inalco, Milano, Italy) for confocal microscopy.

    Molecular Weight:

    Article Title: Stream-related preferences of inputs to the superior colliculus from areas of dorsal and ventral streams of mouse visual cortex
    Article Snippet: To label the corticotectal connections we used the predominantly anterograde tracer, biotinylated dextran amine (BDA; 10,000 molecular weight, 5% in H2 O, Invitrogen). .. The retrograde tracer, bisbenzimide (5% in H2 O, Sigma) was pressure-injected (Picospritzer, III, Parker-Hannafin) through glass pipettes (20 μm tip diameter) at 30–40 sites (20–50 nl each) distributed across the right posterior cortical hemisphere.

    TUNEL Assay:

    Article Title: Inactivation of DNase1L2 and DNase2 in keratinocytes suppresses DNA degradation during epidermal cornification and results in constitutive parakeratosis
    Article Snippet: Paragraph title: TUNEL ... The sections were also subjected to DNA labeling with Hoechst 33258 (Sigma Aldrich, St. Louis, MO).

    In Vivo:

    Article Title: Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy
    Article Snippet: Paragraph title: In vivo labeling with fluorescent probes ... Hoechst 33258 (Sigma, Schnelldorf, Germany) was dissolved in saline and 200 μg was applied intraperitoneally or intravenously.

    Fluorescence:

    Article Title: Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors
    Article Snippet: Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). .. Images were obtained using a fluorescence microscope (Dp Manager 2.1; Olympus Optical Co., Tokyo, Japan).

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: Briefly, MDA-MB-231 cells were stained with 1 µM calcein green AM (Invitrogen Corporation; Carlsbad, CA, USA) and then incubated with the pharmacological blockers (1 µM NTS or 0.5 µM SR 48692, an NTSR1 non-peptide antagonist, or 0.45 M sucrose) 30 min before and during the 30-min incubation with propidium iodide-labeled NTS-polyplex nanoparticles. .. Upon the completion of the assay, cells were counterstained with Hoechst 33258 (Sigma-Aldrich; St. Louis, MO, USA) and analyzed using a laser scanning spectral confocal microscope (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany) to detect the fluorescence at Ex-Em wavelengths of 405–461 nm for Hoechst 33258 (blue channel), 488–517 nm for calcein (green channel), and 532–617 nm for propidium iodide (red channel). .. Ten to twenty consecutive optical sections of 1 µm were taken in a bidimensional plane.

    Multiple Displacement Amplification:

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: Paragraph title: Targeted Gene Delivery to MDA-MB-231 Tumors ... Tumor slices (12 µm) were mounted on glass coverslips and counterstained with fluorescein (FITC)-labeled phalloidin (Invitrogen; Eugene, OR, USA), in the case of internalization assay, or with Hoechst 33258 (Sigma-Aldrich Co.; St. Louis, MO, USA), in the case of expression assays.

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: Briefly, MDA-MB-231 cells were stained with 1 µM calcein green AM (Invitrogen Corporation; Carlsbad, CA, USA) and then incubated with the pharmacological blockers (1 µM NTS or 0.5 µM SR 48692, an NTSR1 non-peptide antagonist, or 0.45 M sucrose) 30 min before and during the 30-min incubation with propidium iodide-labeled NTS-polyplex nanoparticles. .. Upon the completion of the assay, cells were counterstained with Hoechst 33258 (Sigma-Aldrich; St. Louis, MO, USA) and analyzed using a laser scanning spectral confocal microscope (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany) to detect the fluorescence at Ex-Em wavelengths of 405–461 nm for Hoechst 33258 (blue channel), 488–517 nm for calcein (green channel), and 532–617 nm for propidium iodide (red channel).

    Immunohistochemistry:

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons
    Article Snippet: For immunohistochemistry, embryos were fixed for 8 h at 4°C with 4% paraformaldehyde (Merck), cryopreserved ON at 4°C in PBS containing 30% sucrose (Merck), and embedded in the OCT compound Tissue-Tek (Sakura). .. The sections were finally washed 5 times in PBTx, once in PBS, and they were then incubated with 1 µg/ml bisbenzimide (Sigma) in PBS before mounting in glycerol/PBS (1∶1).

    Article Title: Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization
    Article Snippet: Paragraph title: Immunohistochemistry and morphometric analysis ... The nuclei of cells were counterstained with Hoechst 33258 (Sigma).

    Labeling:

    Article Title: Stream-related preferences of inputs to the superior colliculus from areas of dorsal and ventral streams of mouse visual cortex
    Article Snippet: The retrograde tracer, bisbenzimide (5% in H2 O, Sigma) was pressure-injected (Picospritzer, III, Parker-Hannafin) through glass pipettes (20 μm tip diameter) at 30–40 sites (20–50 nl each) distributed across the right posterior cortical hemisphere. .. The retrograde tracer, bisbenzimide (5% in H2 O, Sigma) was pressure-injected (Picospritzer, III, Parker-Hannafin) through glass pipettes (20 μm tip diameter) at 30–40 sites (20–50 nl each) distributed across the right posterior cortical hemisphere.

    Article Title: Human Mesenchymal Stem Cells Prolong Survival and Ameliorate Motor Deficit through Trophic Support in Huntington's Disease Mouse Models
    Article Snippet: The cell-line was maintained in DMEM (GibcoBRL) with 10% FBS (GibcoBRL) and 100 U/ml penicillin/streptomycin (GibcoBRL). .. On the day of transplantation, hBM-MSCs were labeled with bisbenzimide (Bis; 1 µg/ml; Sigma) 10 min before detachment with trypsin. .. Before surgery, 12-week-old male C57/B6 mice were anesthetized (0.5 g/kg chloral hydrate i.p.) and positioned in a stereotaxic apparatus (Stoelting).

    Article Title: Contribution of Schwann Cells to Remyelination in a Naturally Occurring Canine Model of CNS Neuroinflammation
    Article Snippet: In addition, double labeling for P0 and periaxin was performed. .. Nuclear counterstaining was performed with 0.01% bisbenzimide (H33258, Sigma Aldrich, Taufkirchen, Germany) and sections were mounted with Dako Fluorescent Mounting medium (DakoCytomation, Hamburg, Germany).

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons
    Article Snippet: Nuclear labeling was then performed with PBS containing 1 µg/ml bisbenzimide, and the preparations were then mounted in glycerol (Panreac)/PBS (1∶1). .. The sections were finally washed 5 times in PBTx, once in PBS, and they were then incubated with 1 µg/ml bisbenzimide (Sigma) in PBS before mounting in glycerol/PBS (1∶1).

    Article Title: Complex morphology and functional dynamics of vital murine intestinal mucosa revealed by autofluorescence 2-photon microscopy
    Article Snippet: Paragraph title: In vivo labeling with fluorescent probes ... Hoechst 33258 (Sigma, Schnelldorf, Germany) was dissolved in saline and 200 μg was applied intraperitoneally or intravenously.

    Purification:

    Article Title: Influence of DNA Structure on Adjacent Site Cooperative Binding
    Article Snippet: The DNA hairpin, 5′-CTTTTGCAAAAGTCTCCTTTTGCAAAAG-3′, was purchased from Midland Certified Reagent Company with HPLC purification and mass spectrometry characterization. .. The DNA duplex d(CTTTTGCAAAAG)2 was purchased from Integrated DNA Technologies, Inc. Hoechst 33258 was purchased from Sigma-Aldrich, and DB183 was synthesized as previously described.

    Article Title: Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis
    Article Snippet: They were then washed with PBS and permeabilised with 0.5% Triton X-100 in PBS at room temperature for 5 min. After blocking with PBS + 5% normal goat serum for 30 min at room temperature, coverslips were incubated overnight with primary antibodies diluted in PBS + 5% normal goat serum at 4°C: mouse monoclonal antibody against the native form of cytochrome c (1:20; BD Pharmingen #556432), rabbit polyclonal antibody against the cleaved form (Asp175) of caspase 3 (1:500; Cell Signaling Technology #9661) or purified anti-peptide antibody against Trim17 (1:100). .. In the last wash, 1 μg/ml of Hoechst 33258 (Sigma) was added to the cells for 10 min at room temperature.

    Article Title: DNA orientation-specific adhesion and patterning of living mammalian cells on self-assembled DNA monolayers †Electronic supplementary information (ESI) available: Details in experimental section and supporting figures. See DOI: 10.1039/c5sc04102cClick here for additional data file.
    Article Snippet: DNA oligonucleotides were purchased from Takara (purified by HPLC). .. Inc. Mercaptohexanol (MCH), Hoechst 33258, phalloidin–tetramethylrhodamine B isothiocyanate (TRITC) and SMCC were purchased from Sigma.

    Immunostaining:

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons
    Article Snippet: Paragraph title: Immunostaining ... The sections were finally washed 5 times in PBTx, once in PBS, and they were then incubated with 1 µg/ml bisbenzimide (Sigma) in PBS before mounting in glycerol/PBS (1∶1).

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons
    Article Snippet: Cells were counted on a Nikon (Melville, NY) E80i microscope using a 40× objective with phase contrast and epifluorescence illumination, and an average of 100–400 cells were analyzed per coverslip. .. Mitotic nuclei were identified by either DNA staining with 1 µg/ml bisbenzimide (Sigma) or phosphoHistone H3 immunostaining. .. Apoptosis was measured as the percentage of cells showing pyknotic nuclei, as evidenced by bisbenzimide staining.

    Confocal Microscopy:

    Article Title: Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques
    Article Snippet: Samples were rehydrated for 15 min in PBS and then immunolabeled with the antibodies listed in . .. All the incubations were performed at room temperature for 1 h. Cells were then counterstained for DNA with 0.1 µg/mL of Hoechst 33258 (Sigma-Aldrich, Milano, Italy) for 10 min, washed with PBS, and mounted in a drop of Mowiol (Calbiochem-Inalco, Milano, Italy) for confocal microscopy. .. Confocal laser scanning microscopy was performed with a Leica TCS-SP system mounted on a Leica DMIRBE inverted microscope.

    Mouse Assay:

    Article Title: Stream-related preferences of inputs to the superior colliculus from areas of dorsal and ventral streams of mouse visual cortex
    Article Snippet: The retrograde tracer, bisbenzimide (5% in H2 O, Sigma) was pressure-injected (Picospritzer, III, Parker-Hannafin) through glass pipettes (20 μm tip diameter) at 30–40 sites (20–50 nl each) distributed across the right posterior cortical hemisphere. .. The retrograde tracer, bisbenzimide (5% in H2 O, Sigma) was pressure-injected (Picospritzer, III, Parker-Hannafin) through glass pipettes (20 μm tip diameter) at 30–40 sites (20–50 nl each) distributed across the right posterior cortical hemisphere.

    Plasmid Preparation:

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: For the internalization assays, tumors were dissected 6 h after the local injection or 24 h after the intravenous injection of propidium iodide-labeled NTS-polyplex nanoparticles harboring the plasmid pEGFP-N1 , , as described elsewhere . .. Tumor slices (12 µm) were mounted on glass coverslips and counterstained with fluorescein (FITC)-labeled phalloidin (Invitrogen; Eugene, OR, USA), in the case of internalization assay, or with Hoechst 33258 (Sigma-Aldrich Co.; St. Louis, MO, USA), in the case of expression assays.

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: Propidium iodide-labeled NTS-polyplex nanoparticles harboring the plasmid pEGFP-N1 were used for the internalization and pharmacological blocking assays in MDA-MB-231 cells following the procedure that was previously described , , . .. Upon the completion of the assay, cells were counterstained with Hoechst 33258 (Sigma-Aldrich; St. Louis, MO, USA) and analyzed using a laser scanning spectral confocal microscope (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany) to detect the fluorescence at Ex-Em wavelengths of 405–461 nm for Hoechst 33258 (blue channel), 488–517 nm for calcein (green channel), and 532–617 nm for propidium iodide (red channel).

    Software:

    Article Title: Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors
    Article Snippet: Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). .. Images were obtained using a fluorescence microscope (Dp Manager 2.1; Olympus Optical Co., Tokyo, Japan).

    Microscopy:

    Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis
    Article Snippet: Paragraph title: Indirect immunofluorescence and microscopy ... DNA was visualized by staining with Hoechst 33258 (Sigma).

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: Briefly, MDA-MB-231 cells were stained with 1 µM calcein green AM (Invitrogen Corporation; Carlsbad, CA, USA) and then incubated with the pharmacological blockers (1 µM NTS or 0.5 µM SR 48692, an NTSR1 non-peptide antagonist, or 0.45 M sucrose) 30 min before and during the 30-min incubation with propidium iodide-labeled NTS-polyplex nanoparticles. .. Upon the completion of the assay, cells were counterstained with Hoechst 33258 (Sigma-Aldrich; St. Louis, MO, USA) and analyzed using a laser scanning spectral confocal microscope (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany) to detect the fluorescence at Ex-Em wavelengths of 405–461 nm for Hoechst 33258 (blue channel), 488–517 nm for calcein (green channel), and 532–617 nm for propidium iodide (red channel). .. Ten to twenty consecutive optical sections of 1 µm were taken in a bidimensional plane.

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons
    Article Snippet: Cells were counted on a Nikon (Melville, NY) E80i microscope using a 40× objective with phase contrast and epifluorescence illumination, and an average of 100–400 cells were analyzed per coverslip. .. Mitotic nuclei were identified by either DNA staining with 1 µg/ml bisbenzimide (Sigma) or phosphoHistone H3 immunostaining.

    Negative Control:

    Article Title: Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization
    Article Snippet: The nuclei of cells were counterstained with Hoechst 33258 (Sigma). .. The nuclei of cells were counterstained with Hoechst 33258 (Sigma).

    Recombinant:

    Article Title: Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization
    Article Snippet: The nuclei of cells were counterstained with Hoechst 33258 (Sigma). .. For morphometric analysis, serial sections (n=3) of CNV were stained by hematoxylin and eosin, and the degree of staining for EMT-associated transcriptional factors and tissue fibrosis was evaluated based on a 4 grade scale: - (negative), + (10% to 40%), ++ (40% to 70%), or +++ (70% to 100%).

    In Vitro:

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: Paragraph title: Delivery of NTS-polyplex Nanoparticles In vitro ... Upon the completion of the assay, cells were counterstained with Hoechst 33258 (Sigma-Aldrich; St. Louis, MO, USA) and analyzed using a laser scanning spectral confocal microscope (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany) to detect the fluorescence at Ex-Em wavelengths of 405–461 nm for Hoechst 33258 (blue channel), 488–517 nm for calcein (green channel), and 532–617 nm for propidium iodide (red channel).

    Cell Counting:

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons
    Article Snippet: Paragraph title: Cell Counting ... Mitotic nuclei were identified by either DNA staining with 1 µg/ml bisbenzimide (Sigma) or phosphoHistone H3 immunostaining.

    In Situ:

    Article Title: Inactivation of DNase1L2 and DNase2 in keratinocytes suppresses DNA degradation during epidermal cornification and results in constitutive parakeratosis
    Article Snippet: The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Roche Diagnostics GmbH, Vienna, Austria) was performed to label 3′-OH ends of DNA in situ , according to a published protocol . .. The sections were also subjected to DNA labeling with Hoechst 33258 (Sigma Aldrich, St. Louis, MO).

    Marker:

    Article Title: Kainic Acid-Induced Golgi Complex Fragmentation/Dispersal Shifts the Proteolysis of Reelin in Primary Rat Neuronal Cells: An In Vitro Model of Early Stage Epilepsy
    Article Snippet: Primary antibodies, mouse monoclonal anti-Golgi formiminotransferase cyclodeaminase (FTCD, Abcam ab27043, 1/400), rabbit polyclonal anti-cis -Golgi glycoprotein of 130 kDa (GM130, Abcam ab28049, 1/400), rabbit polyclonal anti-ERp29 (Abcam ab11420, 1/500) for detecting the ER marker protein, rabbit polyclonal anti-LAMP (Abcam ab24170, 1/100) for detecting the lysosome marker protein, and mouse monoclonal anti-Reelin (Abcam ab78540, 1/400) were used. .. Finally, cells were washed five times for 10 min in PBST and three times for 5 min in PBS, then processed for Hoechst 33258 (Sigma B2883) for 30 min, washed in PBS, and cover-slipped with Fluoromount.

    Article Title: Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization
    Article Snippet: Subsequently, the sections were reacted with primary antibodies at 4 °C overnight: RPE65 (specific marker for RPE: Transduction Laboratories, Lexington, KY) at a dilution of 1:100, α-smooth muscle actin (SMA; Sigma, St. Louis, MO) at a dilution of 1:500, Snail (Abcam, Cambridge, MA) at a dilution of 1:100, Slug (Abcam) at a dilution of 1:250, Twist (Abcam) at a dilution of 1:650, and SIP1 (Abcam) at a dilution of 1:200. .. The nuclei of cells were counterstained with Hoechst 33258 (Sigma).

    Staining:

    Article Title: Large tumor suppressors 1 and 2 regulate Aurora-B through phosphorylation of INCENP to ensure completion of cytokinesis
    Article Snippet: Briefly, fixed cells were incubated with the indicated primary antibodies, followed by incubation with secondary antibodies [Alexa Fluor 488- or 594-conjugated anti-rabbit/mouse IgG (Molecular Probes, Eugene, OR, USA)] in TBST containing 5% FBS. .. DNA was visualized by staining with Hoechst 33258 (Sigma). .. Cells were observed on a fluorescence microscope (model BX51, Olympus, Tokyo, Japan) or a confocal laser-scanning microscope (model FV10i, Olympus) using the Fluoview software (Olympus).

    Article Title: UV-induced Spectral Shift and Protonation of DNA Fluorescent Dye Hoechst 33258
    Article Snippet: Paragraph title: Cell Staining ... Preparations of fixed cells were rinsed three times with PBS, permeabilised with 70 % ethanol (30 s) and incubated with a solution of Hoechst 33258 [ – ] (2 μg/ml; Sigma-Aldrich, Poland) for 30 min at room temperature (RT).

    Article Title: Adipose-Derived Stem Cell Coculturing Stimulates Integrin-Mediated Extracellular Matrix Adhesion of Melanocytes by Upregulating Growth Factors
    Article Snippet: Paragraph title: Immunofluorescence staining ... Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich).

    Article Title: STAT1-Mediated Down-Regulation of Bcl-2 Expression Is Involved in IFN-γ/TNF-α–Induced Apoptosis in NIT-1 Cells
    Article Snippet: In some experiment, cells were transfected with 60 nM of STAT-1 siRNAs (Santa Cruz) or control siRNA (Santa Cruz) as described above, before IFN-γ and TNF-α treatment. .. Cells were stained with rabbit anti-Bcl-2 antibody (Abcam) or mouse anti-Stat1 (phospho Y701) antibody and then with DyLightTM 488 donkey anti-rabbit IgG (Biolegend) and DyLightTM 488 goat anti-mouse IgG (Biolegend), respectively, followed by 5μg/ml of Hoechst 33258 (Sigma) staining. .. Cells were imaged under a confocal laser scanning microscope (Leica microsystem).

    Article Title: Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice
    Article Snippet: Briefly, MDA-MB-231 cells were stained with 1 µM calcein green AM (Invitrogen Corporation; Carlsbad, CA, USA) and then incubated with the pharmacological blockers (1 µM NTS or 0.5 µM SR 48692, an NTSR1 non-peptide antagonist, or 0.45 M sucrose) 30 min before and during the 30-min incubation with propidium iodide-labeled NTS-polyplex nanoparticles. .. Upon the completion of the assay, cells were counterstained with Hoechst 33258 (Sigma-Aldrich; St. Louis, MO, USA) and analyzed using a laser scanning spectral confocal microscope (Leica TCS SPE; Leica Microsystems, Wetzlar, Germany) to detect the fluorescence at Ex-Em wavelengths of 405–461 nm for Hoechst 33258 (blue channel), 488–517 nm for calcein (green channel), and 532–617 nm for propidium iodide (red channel).

    Article Title: Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis
    Article Snippet: In the last wash, 1 μg/ml of Hoechst 33258 (Sigma) was added to the cells for 10 min at room temperature. .. In the last wash, 1 μg/ml of Hoechst 33258 (Sigma) was added to the cells for 10 min at room temperature.

    Article Title: Brain-Derived Neurotrophic Factor-Dependent cdk1 Inhibition Prevents G2/M Progression in Differentiating Tetraploid Neurons
    Article Snippet: Cells were counted on a Nikon (Melville, NY) E80i microscope using a 40× objective with phase contrast and epifluorescence illumination, and an average of 100–400 cells were analyzed per coverslip. .. Mitotic nuclei were identified by either DNA staining with 1 µg/ml bisbenzimide (Sigma) or phosphoHistone H3 immunostaining. .. Apoptosis was measured as the percentage of cells showing pyknotic nuclei, as evidenced by bisbenzimide staining.

    Article Title: Transcriptional factors associated with epithelial-mesenchymal transition in choroidal neovascularization
    Article Snippet: The nuclei of cells were counterstained with Hoechst 33258 (Sigma). .. The nuclei of cells were counterstained with Hoechst 33258 (Sigma).

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  • 99
    Millipore hoechst 33258
    A great variety of proteins are detected by antibody, GFP-/EYFP-/RFP-fusion constructs, or X-Gal staining for active β-galactosidase produced by  lacZ -containing  P -element insertion stocks. We consistently used 9–10 hr old prepupal salivary glands for these types of detection. ( a ) Salivary gland showing the presence of nuclear receptor E75 (red) and a portion of the cytoplasmic signaling protein Ras2 (green) in the lumen. The cortical membrane is stained with AF 488 -phalloidin for F-actin. ( b ) Similarly to ( a ), two cytoplasmic proteins, Oho-31 (green) and tight junction membrane protein Arm (red) were found secreted into the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). ( c ) Tumor suppressor protein p127, the product of  l(2)gl  gene (green), and the nucleolar component fibrillarin (red) are found secreted in the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). Fluorescently-tagged constructs (most using GFP-), showed that many fusion proteins were secreted into the lumen. These are exemplified by GFP-Rbp1 ( d ). Examples of proteins monitored via  lacZ -fusion include the transcription factor Ttk ( e ), the dual-specific LAMMER kinase Doa ( f ), the D subunit of the vacuolar H +  vATPase Vha36-1 ( g ) and the transcription factor Fkh ( h ).
    Hoechst 33258, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A great variety of proteins are detected by antibody, GFP-/EYFP-/RFP-fusion constructs, or X-Gal staining for active β-galactosidase produced by  lacZ -containing  P -element insertion stocks. We consistently used 9–10 hr old prepupal salivary glands for these types of detection. ( a ) Salivary gland showing the presence of nuclear receptor E75 (red) and a portion of the cytoplasmic signaling protein Ras2 (green) in the lumen. The cortical membrane is stained with AF 488 -phalloidin for F-actin. ( b ) Similarly to ( a ), two cytoplasmic proteins, Oho-31 (green) and tight junction membrane protein Arm (red) were found secreted into the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). ( c ) Tumor suppressor protein p127, the product of  l(2)gl  gene (green), and the nucleolar component fibrillarin (red) are found secreted in the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). Fluorescently-tagged constructs (most using GFP-), showed that many fusion proteins were secreted into the lumen. These are exemplified by GFP-Rbp1 ( d ). Examples of proteins monitored via  lacZ -fusion include the transcription factor Ttk ( e ), the dual-specific LAMMER kinase Doa ( f ), the D subunit of the vacuolar H +  vATPase Vha36-1 ( g ) and the transcription factor Fkh ( h ).

    Journal: PLoS ONE

    Article Title: Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    doi: 10.1371/journal.pone.0094383

    Figure Lengend Snippet: A great variety of proteins are detected by antibody, GFP-/EYFP-/RFP-fusion constructs, or X-Gal staining for active β-galactosidase produced by lacZ -containing P -element insertion stocks. We consistently used 9–10 hr old prepupal salivary glands for these types of detection. ( a ) Salivary gland showing the presence of nuclear receptor E75 (red) and a portion of the cytoplasmic signaling protein Ras2 (green) in the lumen. The cortical membrane is stained with AF 488 -phalloidin for F-actin. ( b ) Similarly to ( a ), two cytoplasmic proteins, Oho-31 (green) and tight junction membrane protein Arm (red) were found secreted into the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). ( c ) Tumor suppressor protein p127, the product of l(2)gl gene (green), and the nucleolar component fibrillarin (red) are found secreted in the lumen; nuclei are stained for DNA with Hoechst 33258 (blue). Fluorescently-tagged constructs (most using GFP-), showed that many fusion proteins were secreted into the lumen. These are exemplified by GFP-Rbp1 ( d ). Examples of proteins monitored via lacZ -fusion include the transcription factor Ttk ( e ), the dual-specific LAMMER kinase Doa ( f ), the D subunit of the vacuolar H + vATPase Vha36-1 ( g ) and the transcription factor Fkh ( h ).

    Article Snippet: Depending on the fluorochrome combination for antibodies and phalloidin, nuclei were counterstained for DNA either with 5 µg/ml Hoechst-33258 (Calbiochem), 0.5 µg/ml Oli-Green or 0.1 µg/ml Toto-3 (both Molecular Probes Inc.).

    Techniques: Construct, Staining, Produced

    Evidence for apocrine secretion of undegraded proteins and the presence of intact genomic DNA in nuclei, and for the release of mitochondria into lumen. Panels a and b show western blots of secreted proteins isolated from the lumen. ( a ) Rab11 protein was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion (lane 3). ( b ) The transcription factor BR-C Z1 was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion from +9–10 hr APF (lane 3). ( c ) In +8–8.5 hr APF prepupae, ribosomal protein Rp40 (green) and β-tubulin (red) are detectable in the lumen of the salivary glands, while the signal for DNA remains nuclear. ( d ) In +9 hr APF prepupae, the ribosomal protein Rp21 (green) and transcription factor E74 (red) are detected in the lumen, while the signal for DNA remains nuclear. ( e ) In +10 hr APF prepupae, both the ribosomal protein p127 (green) and the transcription factor BR-C (red) are detected in the lumen, while the signal for DNA remains nuclear throughout the entire salivary gland, including its columnar, transitional and corpuscular cells; confocal images 80×. ( f ,  g ) Mitochondria are released by apocrine secretion into the lumen as evidenced by chasing a vital Rhodamine 123 signal. In larval as well as early prepupal salivary glands, intact living mitochondria are visible only inside cells ( f ), whereas in +8–10 hr APF prepupae they also can be detected inside the lumen ( g ); both confocal images 630×. This is also consistent with detection of more than dozen of various mitochondrial proteins listed in   Tables 1  through   4 . In addition,  in situ  hybridization with a mitochondrial genome-specific DNA probe (3'-OH end of mt cytochrome c oxidase I, entire coding sequence of mt tRNA-Leu, and 5'-OH end of mt cytochrome c oxidase II) confirmed the presence of mitochondrial DNA in the secretory material in +9 hr APF prepupae ( h ,  i , (green)) along with F-actin ( h ,  j , (blue)). Although nuclear proteins are released by an apocrine mechanism into the lumen, nuclear DNA was never detected in the secretion. When  in situ  hybridization was performed in +9 hr APF prepupae with a probe for a nuclear gene  Doa  locus, signal was found only in nuclei ( k ,  n , (red)) together with Hoechst 33258 staining DNA ( k ,  l , (green)), while F-actin was detectable in the lumen ( k ,  m , (blue)). Remaining confocal images 400×. L in ( f ), ( g ), ( h ) and ( k )  =  lumen.

    Journal: PLoS ONE

    Article Title: Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    doi: 10.1371/journal.pone.0094383

    Figure Lengend Snippet: Evidence for apocrine secretion of undegraded proteins and the presence of intact genomic DNA in nuclei, and for the release of mitochondria into lumen. Panels a and b show western blots of secreted proteins isolated from the lumen. ( a ) Rab11 protein was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion (lane 3). ( b ) The transcription factor BR-C Z1 was detected in total protein extracts from late larval salivary glands (lane 1), +7 hr APF prepupal salivary glands (lane 2), and the isolated luminal secretion from +9–10 hr APF (lane 3). ( c ) In +8–8.5 hr APF prepupae, ribosomal protein Rp40 (green) and β-tubulin (red) are detectable in the lumen of the salivary glands, while the signal for DNA remains nuclear. ( d ) In +9 hr APF prepupae, the ribosomal protein Rp21 (green) and transcription factor E74 (red) are detected in the lumen, while the signal for DNA remains nuclear. ( e ) In +10 hr APF prepupae, both the ribosomal protein p127 (green) and the transcription factor BR-C (red) are detected in the lumen, while the signal for DNA remains nuclear throughout the entire salivary gland, including its columnar, transitional and corpuscular cells; confocal images 80×. ( f , g ) Mitochondria are released by apocrine secretion into the lumen as evidenced by chasing a vital Rhodamine 123 signal. In larval as well as early prepupal salivary glands, intact living mitochondria are visible only inside cells ( f ), whereas in +8–10 hr APF prepupae they also can be detected inside the lumen ( g ); both confocal images 630×. This is also consistent with detection of more than dozen of various mitochondrial proteins listed in Tables 1 through 4 . In addition, in situ hybridization with a mitochondrial genome-specific DNA probe (3'-OH end of mt cytochrome c oxidase I, entire coding sequence of mt tRNA-Leu, and 5'-OH end of mt cytochrome c oxidase II) confirmed the presence of mitochondrial DNA in the secretory material in +9 hr APF prepupae ( h , i , (green)) along with F-actin ( h , j , (blue)). Although nuclear proteins are released by an apocrine mechanism into the lumen, nuclear DNA was never detected in the secretion. When in situ hybridization was performed in +9 hr APF prepupae with a probe for a nuclear gene Doa locus, signal was found only in nuclei ( k , n , (red)) together with Hoechst 33258 staining DNA ( k , l , (green)), while F-actin was detectable in the lumen ( k , m , (blue)). Remaining confocal images 400×. L in ( f ), ( g ), ( h ) and ( k )  =  lumen.

    Article Snippet: Depending on the fluorochrome combination for antibodies and phalloidin, nuclei were counterstained for DNA either with 5 µg/ml Hoechst-33258 (Calbiochem), 0.5 µg/ml Oli-Green or 0.1 µg/ml Toto-3 (both Molecular Probes Inc.).

    Techniques: Western Blot, Isolation, In Situ, Hybridization, Sequencing, Staining

    Evidence for the graded temporal release of different proteins by apocrine secretion. ( a ) = At+8.5 hr APF, the ribosomal protein Rp40 (blue) is completely released into lumen, the cortical membrane component α-spectrin (green) was removed from the lateral and apical surfaces but remained at the basal surface, and the nuclear receptor Usp (red) is about half-released into the lumen. ( b ) At +9 hr APF, both the ribosomal protein Rp21 (green) as well as the ecdysone-inducible Ets-like E74 transcription factor (red) are present only in the lumen, whereas there remains significant F-actin (blue) signal on the cortical membranes. ( c ) At the same time (+9 hr APF), the ecdysone-regulated transcription factor and nuclear tumor suppressor are secreted differently: while Kr-h (red ( d )) is completely extruded into the lumen, p53 (green ( e )) only starts to be released and the majority of its signal is still detected in nuclei. Although filamentous actin (blue ( f )) already is being secreted into the lumen, there is detectable signal still visible on cell membranes. ( g ) During +9 to +10 hr APF, the ecdysone-regulated transcription factor BR-C (green ( h )) is completely released into the lumen, whereas lamin C (red), a component of the nuclear envelope, is only partially released and can be still detected on the nuclear membrane ( i ). Although filamentous actin (blue) is already within the lumen, significant amounts of it still line the cortical cytoskeleton, mainly at the apical membrane ( j ). ( k ) At the end of +10 hr APF both, Rab11 (green ( l )), a member of the GTPase family of membrane proteins as well as the tumor suppressor transcription factor p53 (red ( m )) have been completely secreted into the lumen. Hoechst 33258 was used to detect nuclear DNA (blue ( n )) which stays in nuclei. All confocal images 400×.

    Journal: PLoS ONE

    Article Title: Apocrine Secretion in Drosophila Salivary Glands: Subcellular Origin, Dynamics, and Identification of Secretory Proteins

    doi: 10.1371/journal.pone.0094383

    Figure Lengend Snippet: Evidence for the graded temporal release of different proteins by apocrine secretion. ( a ) = At+8.5 hr APF, the ribosomal protein Rp40 (blue) is completely released into lumen, the cortical membrane component α-spectrin (green) was removed from the lateral and apical surfaces but remained at the basal surface, and the nuclear receptor Usp (red) is about half-released into the lumen. ( b ) At +9 hr APF, both the ribosomal protein Rp21 (green) as well as the ecdysone-inducible Ets-like E74 transcription factor (red) are present only in the lumen, whereas there remains significant F-actin (blue) signal on the cortical membranes. ( c ) At the same time (+9 hr APF), the ecdysone-regulated transcription factor and nuclear tumor suppressor are secreted differently: while Kr-h (red ( d )) is completely extruded into the lumen, p53 (green ( e )) only starts to be released and the majority of its signal is still detected in nuclei. Although filamentous actin (blue ( f )) already is being secreted into the lumen, there is detectable signal still visible on cell membranes. ( g ) During +9 to +10 hr APF, the ecdysone-regulated transcription factor BR-C (green ( h )) is completely released into the lumen, whereas lamin C (red), a component of the nuclear envelope, is only partially released and can be still detected on the nuclear membrane ( i ). Although filamentous actin (blue) is already within the lumen, significant amounts of it still line the cortical cytoskeleton, mainly at the apical membrane ( j ). ( k ) At the end of +10 hr APF both, Rab11 (green ( l )), a member of the GTPase family of membrane proteins as well as the tumor suppressor transcription factor p53 (red ( m )) have been completely secreted into the lumen. Hoechst 33258 was used to detect nuclear DNA (blue ( n )) which stays in nuclei. All confocal images 400×.

    Article Snippet: Depending on the fluorochrome combination for antibodies and phalloidin, nuclei were counterstained for DNA either with 5 µg/ml Hoechst-33258 (Calbiochem), 0.5 µg/ml Oli-Green or 0.1 µg/ml Toto-3 (both Molecular Probes Inc.).

    Techniques: