af647 conjugated secondary antibodies  (Thermo Fisher)


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    Name:
    Goat anti Rabbit IgG H L Highly Cross Adsorbed Secondary Antibody
    Description:
    Goat anti Rabbit IgG H L Highly Cross Adsorbed Secondary Antibody for IF ICC Flow
    Catalog Number:
    a11034
    Price:
    None
    Applications:
    Antibodies and Secondary Detection|Cell Analysis|Secondary Detection
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher af647 conjugated secondary antibodies
    Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with <t>AF647</t> (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).
    Goat anti Rabbit IgG H L Highly Cross Adsorbed Secondary Antibody for IF ICC Flow
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    af647 conjugated secondary antibodies - by Bioz Stars, 2020-02
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    Images

    1) Product Images from "Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process"

    Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

    Journal: Journal of Virology

    doi: 10.1128/JVI.00537-17

    Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).
    Figure Legend Snippet: Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).

    Techniques Used: Infection, Live Cell Imaging, Labeling, Incubation, Western Blot

    L1 proteins that accompany the viral genome into the nucleus dissociate after release of the viral genome. (A) At 24 h, uninfected HaCaT cells were fixed, permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100, and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were incubated with pAb rabbit anti-TGN46 (cyan), which recognizes a luminal epitope of TGN46. Lastly, the cells were permeabilized in 0.5% TX-100, followed by incubation with goat anti-rabbit secondary antibody and subsequent mounting in DAPI (white). Note the lack of reactivity of luminal anti-TGN46 antibody after digitonin treatment. (B and C) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml (B) or 0.5% TX-100 (C), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L1-7 (cyan) for specific detection of the L1 protein and mounted in DAPI (white). (D and E) The percent accessibility of viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible (AC) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L1 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L1 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells, and > 800 EdU puncta were counted per experiment). Mitosis: %IN = 88.58% ± 7.67%, %AC = 11.42% ± 7.67%, %L1 of IN = 82.3% ± 7.30%, %L1 of AC = 42.665% ± 9.335%. Interphase: %IN = 34.53% ± 0.427%, %AC = 68.36% ± 6.463%, %L1 of IN = 58.8% ± 3.805%, %L1 of AC = 20.84% ± 9.159%.
    Figure Legend Snippet: L1 proteins that accompany the viral genome into the nucleus dissociate after release of the viral genome. (A) At 24 h, uninfected HaCaT cells were fixed, permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100, and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were incubated with pAb rabbit anti-TGN46 (cyan), which recognizes a luminal epitope of TGN46. Lastly, the cells were permeabilized in 0.5% TX-100, followed by incubation with goat anti-rabbit secondary antibody and subsequent mounting in DAPI (white). Note the lack of reactivity of luminal anti-TGN46 antibody after digitonin treatment. (B and C) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml (B) or 0.5% TX-100 (C), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L1-7 (cyan) for specific detection of the L1 protein and mounted in DAPI (white). (D and E) The percent accessibility of viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible (AC) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L1 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L1 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells, and > 800 EdU puncta were counted per experiment). Mitosis: %IN = 88.58% ± 7.67%, %AC = 11.42% ± 7.67%, %L1 of IN = 82.3% ± 7.30%, %L1 of AC = 42.665% ± 9.335%. Interphase: %IN = 34.53% ± 0.427%, %AC = 68.36% ± 6.463%, %L1 of IN = 58.8% ± 3.805%, %L1 of AC = 20.84% ± 9.159%.

    Techniques Used: Incubation, Infection, Labeling, Staining

    L2 proteins that accompany the viral genome into the nucleus remain associated with the viral genome. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml of (A) or 0.5% TX-100 (B), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L2-1 (cyan) for specific detection of the L2 protein and mounted in DAPI (white). (C and D) The percent accessibility of the viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible [AC]) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L2 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L2 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells and > 800 EdU puncta counter per experiment). Mitosis: %IN = 83.67% ± 8.435%, %AC = 16.34% ± 8.435%, %L2 of IN = 46.15% ± 6.15%, %L2 of AC = 24.45% ± 16.55%. Interphase: %IN = 42.15% ± 1.35%, %AC = 57.85% ± 1.35%, %L2 of IN = 44.65% ± 6.35%, %L2 of AC = 30.65% ± 3.35%.
    Figure Legend Snippet: L2 proteins that accompany the viral genome into the nucleus remain associated with the viral genome. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml of (A) or 0.5% TX-100 (B), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L2-1 (cyan) for specific detection of the L2 protein and mounted in DAPI (white). (C and D) The percent accessibility of the viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible [AC]) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L2 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L2 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells and > 800 EdU puncta counter per experiment). Mitosis: %IN = 83.67% ± 8.435%, %AC = 16.34% ± 8.435%, %L2 of IN = 46.15% ± 6.15%, %L2 of AC = 24.45% ± 16.55%. Interphase: %IN = 42.15% ± 1.35%, %AC = 57.85% ± 1.35%, %L2 of IN = 44.65% ± 6.35%, %L2 of AC = 30.65% ± 3.35%.

    Techniques Used: Infection, Labeling, Incubation, Staining

    2) Product Images from "The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway"

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aal3322

    Adventitial mvECs in GCA arteries express Jagged1 Tissue biopsies were collected from temporal arteries and from aortic wall specimens. Arteries affected by GCA had typical transmural granulomatous arteritis (GCA-positive and GCA aortitis). Temporal arteries with no inflammatory infiltrates (GCA-negative) served as controls. Nuclei were marked with 4´,6-diamidino-2-phenylindole. Scale bars 50 μm. (A) Tissue-infiltrating T cells were identified by staining sections from GCA-negative and GCA-positive temporal arteries and from GCA aortitis with mouse anti-human CD3 antibody. Antibody binding was visualized with Alexa Fluor 594-labeled anti-mouse immunoglobulin G (IgG) secondary antibody (red). Representative stains from eight samples each are shown here. (B) mRNA was extracted from GCA-positive and GCA-negative temporal arteries and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the expression of JAG1 , DLL1 and DLL4 transcripts. Results (mean ± SEM) from six GCA arteries and six healthy arteries are shown. N.s., not significant. (C and D) Dual-color staining was applied to identify Jagged1 and Delta-like 1 expressed on endothelial cells in GCA-positive, GCA aortitis, and GCA-negative arteries. Tissue sections were double-stained with mouse anti-human CD31 antibody and rabbit anti-human Jagged1 antibody or rabbit anti-human Delta-like 1 antibody. Alexa Fluor 594 anti-mouse IgG (red) and Alexa Fluor 488 anti-rabbit IgG (green) were used as secondary antibodies. Merged images demonstrate colocalization of both markers (yellow). Representative images from eight samples each.
    Figure Legend Snippet: Adventitial mvECs in GCA arteries express Jagged1 Tissue biopsies were collected from temporal arteries and from aortic wall specimens. Arteries affected by GCA had typical transmural granulomatous arteritis (GCA-positive and GCA aortitis). Temporal arteries with no inflammatory infiltrates (GCA-negative) served as controls. Nuclei were marked with 4´,6-diamidino-2-phenylindole. Scale bars 50 μm. (A) Tissue-infiltrating T cells were identified by staining sections from GCA-negative and GCA-positive temporal arteries and from GCA aortitis with mouse anti-human CD3 antibody. Antibody binding was visualized with Alexa Fluor 594-labeled anti-mouse immunoglobulin G (IgG) secondary antibody (red). Representative stains from eight samples each are shown here. (B) mRNA was extracted from GCA-positive and GCA-negative temporal arteries and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the expression of JAG1 , DLL1 and DLL4 transcripts. Results (mean ± SEM) from six GCA arteries and six healthy arteries are shown. N.s., not significant. (C and D) Dual-color staining was applied to identify Jagged1 and Delta-like 1 expressed on endothelial cells in GCA-positive, GCA aortitis, and GCA-negative arteries. Tissue sections were double-stained with mouse anti-human CD31 antibody and rabbit anti-human Jagged1 antibody or rabbit anti-human Delta-like 1 antibody. Alexa Fluor 594 anti-mouse IgG (red) and Alexa Fluor 488 anti-rabbit IgG (green) were used as secondary antibodies. Merged images demonstrate colocalization of both markers (yellow). Representative images from eight samples each.

    Techniques Used: Staining, Binding Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing

    3) Product Images from "Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)"

    Article Title: Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003616

    USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.
    Figure Legend Snippet: USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.

    Techniques Used: Sequencing, Construct, Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    4) Product Images from "A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation"

    Article Title: A Neuron-Optimized CRISPR/dCas9 Activation System for Robust and Specific Gene Regulation

    Journal: eNeuro

    doi: 10.1523/ENEURO.0495-18.2019

    CRISPRa-mediated induction of Fosb is neuron-selective in vivo . A , B , IHC performed for ( A ) NeuN or ( B ) GFAP alongside Fosb demonstrates neuronal selectivity of CRISPRa-mediated Fosb induction. Scale bar = 50 μm. C , Pixel density quantification and cross-correlation analysis reveals a signal overlap between Fosb and NeuN and depletion of signal between Fosb and GFAP ( n = 2 animals with eight regions of interest). All data are expressed as mean ± SEM.
    Figure Legend Snippet: CRISPRa-mediated induction of Fosb is neuron-selective in vivo . A , B , IHC performed for ( A ) NeuN or ( B ) GFAP alongside Fosb demonstrates neuronal selectivity of CRISPRa-mediated Fosb induction. Scale bar = 50 μm. C , Pixel density quantification and cross-correlation analysis reveals a signal overlap between Fosb and NeuN and depletion of signal between Fosb and GFAP ( n = 2 animals with eight regions of interest). All data are expressed as mean ± SEM.

    Techniques Used: In Vivo, Immunohistochemistry

    5) Product Images from "Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network"

    Article Title: Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network

    Journal: eNeuro

    doi: 10.1523/ENEURO.0400-18.2019

    IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p
    Figure Legend Snippet: IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p

    Techniques Used: Immunohistochemistry, Mouse Assay, Labeling

    Dual-label ISH for Pomc and Vglut2 or Gad67 , and relative Pomc expression in the medial basal hypothalamus of control and restored mice. A , ISH for Vglut2 (silver grains) and Pomc (red) in a female control mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to red for these images. B , ISH for Gad67 (silver grains) and Pomc (red) in a female control mouse. C , ISH for Vglut2 (silver grains) and Pomc (red) in a female restored mouse. D , ISH for Gad67 (silver grains) and Pomc (red) in a female restored mouse. In panels A–D , blue arrows indicate overlap between Pomc and the silver grain ( Vglut2 or Gad67 ) signal. E , Degree of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap percentage is connected by the solid black lines. F , Cell count of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap count is connected by the solid black lines. G , Relative qRT-PCR of Pomc expression in the medial-basal hypothalamus of control (blue bar, left), FNΔ2 (red bar, middle), and restored (green bar, right) mice. Male data are represented by filled blue circles and female data by filled pink circles.
    Figure Legend Snippet: Dual-label ISH for Pomc and Vglut2 or Gad67 , and relative Pomc expression in the medial basal hypothalamus of control and restored mice. A , ISH for Vglut2 (silver grains) and Pomc (red) in a female control mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to red for these images. B , ISH for Gad67 (silver grains) and Pomc (red) in a female control mouse. C , ISH for Vglut2 (silver grains) and Pomc (red) in a female restored mouse. D , ISH for Gad67 (silver grains) and Pomc (red) in a female restored mouse. In panels A–D , blue arrows indicate overlap between Pomc and the silver grain ( Vglut2 or Gad67 ) signal. E , Degree of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap percentage is connected by the solid black lines. F , Cell count of overlap between Pomc and Vglut2 (white bars) or Gad67 (grey bars) in control and restored mice, each animal’s Vlgut2/Pomc and Gad67/Pomc overlap count is connected by the solid black lines. G , Relative qRT-PCR of Pomc expression in the medial-basal hypothalamus of control (blue bar, left), FNΔ2 (red bar, middle), and restored (green bar, right) mice. Male data are represented by filled blue circles and female data by filled pink circles.

    Techniques Used: In Situ Hybridization, Expressing, Mouse Assay, Fluorescence, Cell Counting, Quantitative RT-PCR

    Triple-label ISH for Pomc ( A , E , I ), Gad67 ( B , F , J ), Vglut2 (C,G,K), and overlaid signals ( D , H , L ) in WT mice throughout the rostral-caudal ARC axis. A–D , Low-magnification image of ISH signal for Pomc , Gad67 , Vglut2 , and the overlay of all signals from a male mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to blue for these images. E–H , 40× images from the rostral ARC from a male mouse with Pomc neuron profiles outlined in yellow. I–L , 40× images from the caudal ARC from a female mouse with Pomc neuron profiles outlined in yellow. V indicates Vglut2 + Pomc neurons, G indicates Gad67 + Pomc neurons, and VG indicates Vglut2/Gad67 + Pomc neurons. M , The distribution of Pomc neurons along the rostral-caudal ARC axis. N , The overall percentages of Pomc -only (blue bar with filled inverted triangles), Gad67 + (red bar with filled squares), Vglut2 + (grey bar with filled circles), and Vglut2/Gad67 + (purple bar with filled triangles) Pomc neurons in the arcuate nucleus. Male data are represented by filled blue symbols and female data by filled pink symbols. O , The percentages of Pomc -only (blue line with filled inverted triangles), Gad67 + (red line with filled squares), Vglut2 + (grey line with filled circles), and Vglut2/Gad67 + (purple line with filled triangles) Pomc neurons at each coronal level 1 to 5 along the rostral-caudal ARC axis. P , Linear regression analysis of the relative percentage of each phenotypic category of Pomc neurons along the rostral-caudal ARC axis [ Pomc -only solid blue line, Gad67 + dotted red line, Gad67 + (levels 2–5) solid red line, Vglut2 + solid grey line, Vglut2/Gad67 + solid purple line].
    Figure Legend Snippet: Triple-label ISH for Pomc ( A , E , I ), Gad67 ( B , F , J ), Vglut2 (C,G,K), and overlaid signals ( D , H , L ) in WT mice throughout the rostral-caudal ARC axis. A–D , Low-magnification image of ISH signal for Pomc , Gad67 , Vglut2 , and the overlay of all signals from a male mouse. Note that the green fluorescence of Alexa Fluor 488 used to detect Pomc was pseudocolored to blue for these images. E–H , 40× images from the rostral ARC from a male mouse with Pomc neuron profiles outlined in yellow. I–L , 40× images from the caudal ARC from a female mouse with Pomc neuron profiles outlined in yellow. V indicates Vglut2 + Pomc neurons, G indicates Gad67 + Pomc neurons, and VG indicates Vglut2/Gad67 + Pomc neurons. M , The distribution of Pomc neurons along the rostral-caudal ARC axis. N , The overall percentages of Pomc -only (blue bar with filled inverted triangles), Gad67 + (red bar with filled squares), Vglut2 + (grey bar with filled circles), and Vglut2/Gad67 + (purple bar with filled triangles) Pomc neurons in the arcuate nucleus. Male data are represented by filled blue symbols and female data by filled pink symbols. O , The percentages of Pomc -only (blue line with filled inverted triangles), Gad67 + (red line with filled squares), Vglut2 + (grey line with filled circles), and Vglut2/Gad67 + (purple line with filled triangles) Pomc neurons at each coronal level 1 to 5 along the rostral-caudal ARC axis. P , Linear regression analysis of the relative percentage of each phenotypic category of Pomc neurons along the rostral-caudal ARC axis [ Pomc -only solid blue line, Gad67 + dotted red line, Gad67 + (levels 2–5) solid red line, Vglut2 + solid grey line, Vglut2/Gad67 + solid purple line].

    Techniques Used: In Situ Hybridization, Mouse Assay, Fluorescence

    6) Product Images from "Glycosylation of the Hemagglutinin Protein of H5N1 Influenza Virus Increases Its Virulence in Mice by Exacerbating the Host Immune Response"

    Article Title: Glycosylation of the Hemagglutinin Protein of H5N1 Influenza Virus Increases Its Virulence in Mice by Exacerbating the Host Immune Response

    Journal: Journal of Virology

    doi: 10.1128/JVI.02215-16

    Expression of viral proteins in infected MDCK cells. MDCK cells were infected with viruses at an MOI of 5. The infected cells were collected at 12 h postinfection, lysed in SDS loading buffer, and further analyzed by Western blotting (A and B). (A) Protein bands of HA, NP, and M1 proteins detected by Western blotting. The intensity of each band was measured by using ImageJ software, and relative intensity ratios of HA, NP, and M1 compared with that of GAPDH were calculated (B). The infected cells were digested with trypsin to obtain a single cell suspension at 12 h postinfection. The samples were stained with rabbit monoclonal antibody to influenza A virus H5N1 HA protein and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody and then detected on a FACSAria II (BD Biosciences). (C) Mean fluorescence intensity of HA protein was analyzed with FlowJo X 10.0.7r2 (Tree Star, San Carlos, CA). Data are presented as means ± SD ( n = 3). **, P
    Figure Legend Snippet: Expression of viral proteins in infected MDCK cells. MDCK cells were infected with viruses at an MOI of 5. The infected cells were collected at 12 h postinfection, lysed in SDS loading buffer, and further analyzed by Western blotting (A and B). (A) Protein bands of HA, NP, and M1 proteins detected by Western blotting. The intensity of each band was measured by using ImageJ software, and relative intensity ratios of HA, NP, and M1 compared with that of GAPDH were calculated (B). The infected cells were digested with trypsin to obtain a single cell suspension at 12 h postinfection. The samples were stained with rabbit monoclonal antibody to influenza A virus H5N1 HA protein and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) secondary antibody and then detected on a FACSAria II (BD Biosciences). (C) Mean fluorescence intensity of HA protein was analyzed with FlowJo X 10.0.7r2 (Tree Star, San Carlos, CA). Data are presented as means ± SD ( n = 3). **, P

    Techniques Used: Expressing, Infection, Western Blot, Software, Staining, Fluorescence

    7) Product Images from "Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)"

    Article Title: Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003616

    USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.
    Figure Legend Snippet: USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.

    Techniques Used: Sequencing, Construct, Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    8) Product Images from "TRIM29 promotes DNA virus infections by inhibiting innate immune response"

    Article Title: TRIM29 promotes DNA virus infections by inhibiting innate immune response

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00101-w

    TRIM29 binds and colocalizes with STING in the cytosol. a Immunoblot analysis of endogenous proteins of TRIM29 and STING precipitated with anti-STING or immunoglobulin G (IgG; control) from whole-cell lysates of BMDCs from WT mice stimulated without (No) or with HSV-60 (2.5 μg/ml) for 8 h, and then with MG132 treatment for 3 h. Input, 20% of the BMDCs lysate. b Full-length STING and serial truncations of STING with deletion of various domains ( top ). Immunoblot analysis of purified HA-tagged TRIM29 with anti-HA ( bottom blot ), and immunoblot analysis of purified HA-tagged full-length TRIM29 with anti-HA ( middle blot ) or purified Myc-tagged full-length STING and STING truncation mutants alone with anti-Myc ( top blot ) after incubation with Myc-tagged full-length STING and STING truncation mutants and immunoprecipitation with anti-Myc ( top and middle blots ). c Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains ( left margin ); numbers at ends indicate amino-acid positions ( top ). Below , immunoblot analysis of purified Myc-tagged STING with anti-Myc ( bottom blot ), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone ( top blot ) or after incubation with Myc-tagged STING and immunoprecipitation with anti-Myc ( middle blot ). d Colocalization of endogenous TRIM29 and STING in BEAS-2B cells. Confocal microscopy of BEAS-2B cells without stimulation, or stimulated with dsDNA HSV-60 for 4 h. STING was stained with Rabbit anti-STING polyclonal antibody (Cat: 19851-1-AP, Proteintech), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody ( green ), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 594 goat anti-mouse secondary antibody ( red ). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker ( blue ). Scale bars represent 20 µm. The position of protein markers (shown in kDa) is indicated on the right . Data are representative of three independent experiments
    Figure Legend Snippet: TRIM29 binds and colocalizes with STING in the cytosol. a Immunoblot analysis of endogenous proteins of TRIM29 and STING precipitated with anti-STING or immunoglobulin G (IgG; control) from whole-cell lysates of BMDCs from WT mice stimulated without (No) or with HSV-60 (2.5 μg/ml) for 8 h, and then with MG132 treatment for 3 h. Input, 20% of the BMDCs lysate. b Full-length STING and serial truncations of STING with deletion of various domains ( top ). Immunoblot analysis of purified HA-tagged TRIM29 with anti-HA ( bottom blot ), and immunoblot analysis of purified HA-tagged full-length TRIM29 with anti-HA ( middle blot ) or purified Myc-tagged full-length STING and STING truncation mutants alone with anti-Myc ( top blot ) after incubation with Myc-tagged full-length STING and STING truncation mutants and immunoprecipitation with anti-Myc ( top and middle blots ). c Full-length TRIM29 and serial truncations of TRIM29 with deletion (Δ) of various domains ( left margin ); numbers at ends indicate amino-acid positions ( top ). Below , immunoblot analysis of purified Myc-tagged STING with anti-Myc ( bottom blot ), and immunoblot analysis (with anti-HA) of purified HA-tagged full-length TRIM29 and TRIM29 truncation mutants alone ( top blot ) or after incubation with Myc-tagged STING and immunoprecipitation with anti-Myc ( middle blot ). d Colocalization of endogenous TRIM29 and STING in BEAS-2B cells. Confocal microscopy of BEAS-2B cells without stimulation, or stimulated with dsDNA HSV-60 for 4 h. STING was stained with Rabbit anti-STING polyclonal antibody (Cat: 19851-1-AP, Proteintech), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody ( green ), while TRIM29 was stained with mouse anti-TRIM29 monoclonal antibody (sc-166707, Santa Cruz), followed by Alexa Fluor 594 goat anti-mouse secondary antibody ( red ). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker ( blue ). Scale bars represent 20 µm. The position of protein markers (shown in kDa) is indicated on the right . Data are representative of three independent experiments

    Techniques Used: Mouse Assay, Purification, Incubation, Immunoprecipitation, Confocal Microscopy, Staining, Marker

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    Transduction:

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    Immunostaining:

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    Incubation:

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    Article Snippet: .. The dishes were washed twice with PBS for 5 min. Then, 50 μL of a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (A-11034; Invitrogen) in PBS/0.1% BSA/1 μg/mL DAPI was added to the spot and incubated for 30 min in the dark. .. Dishes were washed twice as above and air-dried; DABCO glycerol mountant (290734; Sigma) or Fluoromount (F4680; Sigma) was then added to the spot, and the samples then sealed with a coverslip for viewing by fluorescence microscopy with a 100× objective.

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    Article Title: Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning
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    Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress
    Article Snippet: After successive washes, cells were then incubated with Alexa Fluor 488 rabbit anti-mouse IgG #A11059 (Life technologies Eugene, OR. .. USA) or Alexa Fluor 488 goat anti-rabbit IgG #A11034 or Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.

    Article Title: Novel peptide probes to assess the tensional state of fibronectin fibers in cancer
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    Activity Assay:

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    Expressing:

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway
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    Western Blot:

    Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process
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    Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137
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    TUNEL Assay:

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    Article Snippet: .. Immunofluorescence Surgical sections of bone tissue were stained using a MEBSTAIN Apoptosis TUNEL Kit Direct (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan), or stained with anti-single standard DNA (ssDNA) Rabbit IgG (#18731 1:50; IBL, Gunma, Japan) followed by Alexa488-conjugated goat anti-rabbit Ig’ (#A-11034 1:200; Invitrogen, Carlsbad, CA). .. Sections were also stained with Alexa Fluor 488-conjugated rat anti-mouse F4/80 (#B116640 1:100 BioLegend, San Diego, CA, USA) and goat anti-mouse TNFα (#K2911 1:100 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by Alexa488-conjugated donkey anti-rat Ig’ (#A-21208 1:200; Invitrogen, Carlsbad, CA) and Alexa568-conjugated donkey anti-goat Ig’ (#A-11057 1:200; Invitrogen, Carlsbad, CA).

    Immunohistochemistry:

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    Infection:

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    Article Snippet: Immunohistochemistry Mock and JEV infected mice were sacrificed (2 days after infection) and their brains were excised. .. The sections were washed and incubated with Alexa 488 conjugated secondary antibody (A 11034, Life technologies) for 1 hour and washed.

    Light Microscopy:

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    Article Snippet: Mounted sections were counterstained with 0.1% cresyl violet acetate (where indicated), coverslipped, and visualized through a light microscope. .. Sections were then washed in PBS followed by the addition of DAPI (Thermo Fisher Scientific #R37605) and the respective secondary antibody: goat anti-rat Alexa Fluor 555 for HA (1:1000, Invitrogen #A-21434), goat anti-mouse Alexa Fluor 488 for CaMKIIα (1:000, Invitrogen #A-11001), and goat anti-rabbit Alexa Fluor 488 for Pvalb (1:1000, Invitrogen #A-11034).

    Generated:

    Article Title: Autophagy is required for ectoplasmic specialization assembly in sertoli cells
    Article Snippet: The VCP (valosin containing protein) polyclonal antibodies were generated in rabbit by using the corresponding recombinant proteins as antigen. .. The Alexa Fluor 680-conjugated goat anti-mouse , and Alexa Fluor 680-conjugated goat anti-rabbit (A21109) for immunoblotting were purchased from Invitrogen.

    other:

    Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress
    Article Snippet: Antibodies used for study VCP #2649, Bip #3183, CHOP #2895, eIF2α #9722 and p-eIF2α #9721, IRE1α #3294, PERK #5683, JNK #9258, p-JNK 4668, p38 #8690, P-p38 #4511, LC3 #3868, Cleaved Caspase3 #9664, Calnexin #2679, PDI #3501, E-cadherin #3195, Vimentin #5741, Zeb1 #3396, Claudin1 #4933, Snail #3879, Slug #9585, Integrin β3 #4702, Akt #9272, P-Akt #9271, mTOR #2972, P-mTOR #2971 (Cell Signaling Technologies Inc. Danvers, MA 01923); GAPDH #FL335 (Santa Cruz); Alexa Fluor 488 goat anti-rabbit IgG #A11034 and Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.

    Article Title: Ubiquilin1 Represses Migration and Epithelial to Mesenchymal Transition of Human Non-small Cell Lung Cancer Cells
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    Article Title: Induction of Stress Granule-Like Structures in Vesicular Stomatitis Virus-Infected Cells
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    Affinity Purification:

    Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137
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    Recombinant:

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    Article Snippet: The VCP (valosin containing protein) polyclonal antibodies were generated in rabbit by using the corresponding recombinant proteins as antigen. .. The Alexa Fluor 680-conjugated goat anti-mouse , and Alexa Fluor 680-conjugated goat anti-rabbit (A21109) for immunoblotting were purchased from Invitrogen.

    Immunofluorescence:

    Article Title: Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function
    Article Snippet: Paragraph title: IFA with PfEMP1 Antibodies on the Bound IEs. ... The dishes were washed twice with PBS for 5 min. Then, 50 μL of a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (A-11034; Invitrogen) in PBS/0.1% BSA/1 μg/mL DAPI was added to the spot and incubated for 30 min in the dark.

    Article Title: Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning
    Article Snippet: For immunofluorescence staining, 30 μM sections were permeabilized and blocked as before and then incubated at 4°C overnight using the following primary antibodies: HA (1:500, Roche Clone 3F10), CaMKIIα (1:1000, Santa Cruz #sc-32288), Gfap alexafluor 488-conjugated (1:1000, Millipore #MAB3402X), and Pvalb (1:1000, Abcam #ab11427). .. Sections were then washed in PBS followed by the addition of DAPI (Thermo Fisher Scientific #R37605) and the respective secondary antibody: goat anti-rat Alexa Fluor 555 for HA (1:1000, Invitrogen #A-21434), goat anti-mouse Alexa Fluor 488 for CaMKIIα (1:000, Invitrogen #A-11001), and goat anti-rabbit Alexa Fluor 488 for Pvalb (1:1000, Invitrogen #A-11034).

    Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress
    Article Snippet: Paragraph title: Immunofluorescence staining ... USA) or Alexa Fluor 488 goat anti-rabbit IgG #A11034 or Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway
    Article Snippet: Paragraph title: Immunofluorescence and immunohistochemistry ... Antibody binding was visualized with Alexa Fluor 594 anti-mouse IgG (1:200; A-11032, Thermo Fisher Scientific) and Alexa Fluor 488 anti-rabbit IgG (1:200; A-11034, Thermo Fisher Scientific) as secondary antibodies, or developed with the VECTASTAIN ABC kit (Vector Laboratories) plus DAB (3,3´-diaminobenzidine) substrate (Vector Laboratories).

    Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137
    Article Snippet: .. Secondary antibodies for immunofluorescence staining include Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Invitrogen, A-11034) and Alexa Fluor® 546-conjugated goat anti-mouse IgG (Invitrogen, A-11030). .. Agarose conjugated with secondary anti-mouse IgG (A6531) and EZview Red affinity gel conjugated with rabbit anti-c-Myc antibody (E6654) were purchased from Sigma.

    Article Title: Elevation of pro-inflammatory cytokine levels following anti-resorptive drug treatment is required for osteonecrosis development in infectious osteomyelitis
    Article Snippet: .. Immunofluorescence Surgical sections of bone tissue were stained using a MEBSTAIN Apoptosis TUNEL Kit Direct (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan), or stained with anti-single standard DNA (ssDNA) Rabbit IgG (#18731 1:50; IBL, Gunma, Japan) followed by Alexa488-conjugated goat anti-rabbit Ig’ (#A-11034 1:200; Invitrogen, Carlsbad, CA). .. Sections were also stained with Alexa Fluor 488-conjugated rat anti-mouse F4/80 (#B116640 1:100 BioLegend, San Diego, CA, USA) and goat anti-mouse TNFα (#K2911 1:100 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by Alexa488-conjugated donkey anti-rat Ig’ (#A-21208 1:200; Invitrogen, Carlsbad, CA) and Alexa568-conjugated donkey anti-goat Ig’ (#A-11057 1:200; Invitrogen, Carlsbad, CA).

    Intravital Microscopy:

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    Article Snippet: These doses were selected as to induce a hemorrhage in the cremaster muscle of similar intensity to that described previously by intravital microscopy for the PI SVMP BaP1 [ ]. .. After three washes in PBS, tissues were incubated for 4 h at 4°C with goat anti-rabbit polyclonal antibody labeled with Alexa Fluor 488 at a dilution of 1:200 (Invitrogen A11034).

    Fluorescence:

    Article Title: Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function
    Article Snippet: The dishes were washed twice with PBS for 5 min. Then, 50 μL of a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (A-11034; Invitrogen) in PBS/0.1% BSA/1 μg/mL DAPI was added to the spot and incubated for 30 min in the dark. .. Dishes were washed twice as above and air-dried; DABCO glycerol mountant (290734; Sigma) or Fluoromount (F4680; Sigma) was then added to the spot, and the samples then sealed with a coverslip for viewing by fluorescence microscopy with a 100× objective.

    Transfection:

    Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress
    Article Snippet: After 72 hrs of transfection or with 48hrs EerI treatment, cells were fixed with 4.0% paraformaldehyde in PBS for 30 min and then permeabilized with 0.2% Triton X-100 for 15 min at room temperature. .. USA) or Alexa Fluor 488 goat anti-rabbit IgG #A11034 or Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.

    Labeling:

    Article Title: Tissue Localization and Extracellular Matrix Degradation by PI, PII and PIII Snake Venom Metalloproteinases: Clues on the Mechanisms of Venom-Induced Hemorrhage
    Article Snippet: .. After three washes in PBS, tissues were incubated for 4 h at 4°C with goat anti-rabbit polyclonal antibody labeled with Alexa Fluor 488 at a dilution of 1:200 (Invitrogen A11034). .. In order to ascertain whether labeling of SVMPs had an effect on their enzymatic activity, proteolytic activity of the labeled enzymes was quantified on gelatin with the EnzChek Gelatinase Assay Kit (Molecular Probes E-12055) at different times during 24 h. Immunostained tissues were mounted on glass slides and visualized using a Zeiss LSM 5 Pascal laser-scanning confocal microscope (Carl Zeiss Ltd) incorporating a 10X objective (numerical aperture 0.3) and 63X oil objective (numerical aperture 1.4).

    Mouse Assay:

    Article Title: Japanese Encephalitis Virus exploits the microRNA-432 to regulate the expression of Suppressor of Cytokine Signaling (SOCS) 5
    Article Snippet: Immunohistochemistry Mock and JEV infected mice were sacrificed (2 days after infection) and their brains were excised. .. The sections were washed and incubated with Alexa 488 conjugated secondary antibody (A 11034, Life technologies) for 1 hour and washed.

    Blocking Assay:

    Article Title: Tissue Localization and Extracellular Matrix Degradation by PI, PII and PIII Snake Venom Metalloproteinases: Clues on the Mechanisms of Venom-Induced Hemorrhage
    Article Snippet: Fixed whole tissues were incubated for 4 h at room temperature in blocking and permeabilization solution (12.5% goat serum, 12.5% fetal bovine serum and 0.5% Triton X-100 in PBS). .. After three washes in PBS, tissues were incubated for 4 h at 4°C with goat anti-rabbit polyclonal antibody labeled with Alexa Fluor 488 at a dilution of 1:200 (Invitrogen A11034).

    Purification:

    Article Title: Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function
    Article Snippet: Binding assays were carried out as above, and after washing off unbound cells, 25 μg/mL purified polyclonal IgG from a rabbit immunized with NTS-DBLα1 of HB3var03 , IT4var07 , IT4var19 , or IgG from a nonimmunized rabbit and diluted in PBS/1% BSA was added to the spot and incubated for 30 min. .. The dishes were washed twice with PBS for 5 min. Then, 50 μL of a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (A-11034; Invitrogen) in PBS/0.1% BSA/1 μg/mL DAPI was added to the spot and incubated for 30 min in the dark.

    Article Title: Elevation of pro-inflammatory cytokine levels following anti-resorptive drug treatment is required for osteonecrosis development in infectious osteomyelitis
    Article Snippet: Immunofluorescence Surgical sections of bone tissue were stained using a MEBSTAIN Apoptosis TUNEL Kit Direct (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan), or stained with anti-single standard DNA (ssDNA) Rabbit IgG (#18731 1:50; IBL, Gunma, Japan) followed by Alexa488-conjugated goat anti-rabbit Ig’ (#A-11034 1:200; Invitrogen, Carlsbad, CA). .. Sections were also stained with purified mouse anti-PCNA (#610664 1:100; BD Transduction Laboratories, Franklin Lakes, New Jersey) followed by Alexa488-conjugated goat anti-mouse Ig’ (#A-11029 1:200; Invitrogen, Carlsbad, CA).

    Plasmid Preparation:

    Article Title: Japanese Encephalitis Virus exploits the microRNA-432 to regulate the expression of Suppressor of Cytokine Signaling (SOCS) 5
    Article Snippet: Antigen unmasking of sections was performed by boiling the sections dipped in Vector antigen unmasking solution (#H-3300, Vector Laboratories). .. The sections were washed and incubated with Alexa 488 conjugated secondary antibody (A 11034, Life technologies) for 1 hour and washed.

    Software:

    Article Title: Tissue Localization and Extracellular Matrix Degradation by PI, PII and PIII Snake Venom Metalloproteinases: Clues on the Mechanisms of Venom-Induced Hemorrhage
    Article Snippet: After three washes in PBS, tissues were incubated for 4 h at 4°C with goat anti-rabbit polyclonal antibody labeled with Alexa Fluor 488 at a dilution of 1:200 (Invitrogen A11034). .. Three-dimensional reconstitution of the images and analysis of co-localization of the SVMPs with collagen IV were carried out using IMARIS x64 7.4.2 image analysis software, which employs the approach developed by Costes et al. [ ].

    Microscopy:

    Article Title: Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function
    Article Snippet: The dishes were washed twice with PBS for 5 min. Then, 50 μL of a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (A-11034; Invitrogen) in PBS/0.1% BSA/1 μg/mL DAPI was added to the spot and incubated for 30 min in the dark. .. Dishes were washed twice as above and air-dried; DABCO glycerol mountant (290734; Sigma) or Fluoromount (F4680; Sigma) was then added to the spot, and the samples then sealed with a coverslip for viewing by fluorescence microscopy with a 100× objective.

    Article Title: Japanese Encephalitis Virus exploits the microRNA-432 to regulate the expression of Suppressor of Cytokine Signaling (SOCS) 5
    Article Snippet: The sections were washed and incubated with Alexa 488 conjugated secondary antibody (A 11034, Life technologies) for 1 hour and washed. .. The sections were then mounted with mounting medium with DAPI (H-1200, Vector Laboratories) and viewed under florescent microscope (Zeiss Axio imager).

    Article Title: Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning
    Article Snippet: Sections were then washed in PBS followed by the addition of DAPI (Thermo Fisher Scientific #R37605) and the respective secondary antibody: goat anti-rat Alexa Fluor 555 for HA (1:1000, Invitrogen #A-21434), goat anti-mouse Alexa Fluor 488 for CaMKIIα (1:000, Invitrogen #A-11001), and goat anti-rabbit Alexa Fluor 488 for Pvalb (1:1000, Invitrogen #A-11034). .. Images were collected using a Leica Widefield Microscope.

    Article Title: Novel peptide probes to assess the tensional state of fibronectin fibers in cancer
    Article Snippet: Primary antibody solution was removed and samples were washed before incubation with secondary goat anti-rabbit Alexa-633 (A-21070, Invitrogen, 1:100 dilution) or goat anti rat Alexa 546 (A-11081, Invitrogen, 1:100 dilution) or goat anti-rabbit Alexa-488 (A-11034, Invitrogen, 1:100 dilution) antibody solution for 45 min. After another washing step, samples were mounted using Prolong Antifade Gold with or without DAPI (Invitrogen). .. The overview mosaic image in Fig. was acquired using a Nikon TE2000-E epifluorescence microscope equipped with a 10× air objective.

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway
    Article Snippet: Antibody binding was visualized with Alexa Fluor 594 anti-mouse IgG (1:200; A-11032, Thermo Fisher Scientific) and Alexa Fluor 488 anti-rabbit IgG (1:200; A-11034, Thermo Fisher Scientific) as secondary antibodies, or developed with the VECTASTAIN ABC kit (Vector Laboratories) plus DAB (3,3´-diaminobenzidine) substrate (Vector Laboratories). .. All sections were analyzed using a confocal microscope system (Carl Zeiss).

    Article Title: Tissue Localization and Extracellular Matrix Degradation by PI, PII and PIII Snake Venom Metalloproteinases: Clues on the Mechanisms of Venom-Induced Hemorrhage
    Article Snippet: After three washes in PBS, tissues were incubated for 4 h at 4°C with goat anti-rabbit polyclonal antibody labeled with Alexa Fluor 488 at a dilution of 1:200 (Invitrogen A11034). .. In order to ascertain whether labeling of SVMPs had an effect on their enzymatic activity, proteolytic activity of the labeled enzymes was quantified on gelatin with the EnzChek Gelatinase Assay Kit (Molecular Probes E-12055) at different times during 24 h. Immunostained tissues were mounted on glass slides and visualized using a Zeiss LSM 5 Pascal laser-scanning confocal microscope (Carl Zeiss Ltd) incorporating a 10X objective (numerical aperture 0.3) and 63X oil objective (numerical aperture 1.4).

    Binding Assay:

    Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process
    Article Snippet: The binding site includes—but is not restricted to—the N-terminal portion of the FG loop (HPV16 L1 residues 260 to 270) ( , , ). .. Primary antibodies were detected using pAb goat anti-rabbit or anti-mouse AF488 or AF647 conjugated secondary antibodies (A-11034, A-11029, A-21244, and A-21236; Thermo Fisher Scientific).

    Article Title: Infected erythrocytes expressing DC13 PfEMP1 differ from recombinant proteins in EPCR-binding function
    Article Snippet: Binding assays were carried out as above, and after washing off unbound cells, 25 μg/mL purified polyclonal IgG from a rabbit immunized with NTS-DBLα1 of HB3var03 , IT4var07 , IT4var19 , or IgG from a nonimmunized rabbit and diluted in PBS/1% BSA was added to the spot and incubated for 30 min. .. The dishes were washed twice with PBS for 5 min. Then, 50 μL of a 1:500 dilution of Alexa Fluor 488 goat anti-rabbit IgG (A-11034; Invitrogen) in PBS/0.1% BSA/1 μg/mL DAPI was added to the spot and incubated for 30 min in the dark.

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway
    Article Snippet: .. Antibody binding was visualized with Alexa Fluor 594 anti-mouse IgG (1:200; A-11032, Thermo Fisher Scientific) and Alexa Fluor 488 anti-rabbit IgG (1:200; A-11034, Thermo Fisher Scientific) as secondary antibodies, or developed with the VECTASTAIN ABC kit (Vector Laboratories) plus DAB (3,3´-diaminobenzidine) substrate (Vector Laboratories). .. All sections were analyzed using a confocal microscope system (Carl Zeiss).

    Next-Generation Sequencing:

    Article Title: Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning
    Article Snippet: The following incubation steps were then performed with intervening PBS washes (3 × 5 min each): HA antibody (1:500, Roche Clone 3F10) in 2% NGS and 0.3% Triton X-100 overnight at 4°C, biotinylated goat anti-rat antibody (1:1000, Jackson ImmunoResearch #112-065-003) in PBST for 2 h at RT, Vectastain ABC solution (Vector Laboratories #PK-4000) for 1.5 h at RT, DAB solution (0.2 mg/ml) containing 0.005% H2 O2 for 20 min at RT. .. Sections were then washed in PBS followed by the addition of DAPI (Thermo Fisher Scientific #R37605) and the respective secondary antibody: goat anti-rat Alexa Fluor 555 for HA (1:1000, Invitrogen #A-21434), goat anti-mouse Alexa Fluor 488 for CaMKIIα (1:000, Invitrogen #A-11001), and goat anti-rabbit Alexa Fluor 488 for Pvalb (1:1000, Invitrogen #A-11034).

    Staining:

    Article Title: Characterization of a Novel Chromatin Sorting Tool Reveals Importance of Histone Variant H3.3 in Contextual Fear Memory and Motor Learning
    Article Snippet: For immunofluorescence staining, 30 μM sections were permeabilized and blocked as before and then incubated at 4°C overnight using the following primary antibodies: HA (1:500, Roche Clone 3F10), CaMKIIα (1:1000, Santa Cruz #sc-32288), Gfap alexafluor 488-conjugated (1:1000, Millipore #MAB3402X), and Pvalb (1:1000, Abcam #ab11427). .. Sections were then washed in PBS followed by the addition of DAPI (Thermo Fisher Scientific #R37605) and the respective secondary antibody: goat anti-rat Alexa Fluor 555 for HA (1:1000, Invitrogen #A-21434), goat anti-mouse Alexa Fluor 488 for CaMKIIα (1:000, Invitrogen #A-11001), and goat anti-rabbit Alexa Fluor 488 for Pvalb (1:1000, Invitrogen #A-11034).

    Article Title: Regulation of VCP/p97 demonstrates the critical balance between cell death and epithelial-mesenchymal transition (EMT) downstream of ER stress
    Article Snippet: Paragraph title: Immunofluorescence staining ... USA) or Alexa Fluor 488 goat anti-rabbit IgG #A11034 or Alexa Fluor 546 goat anti-rabbit IgG #A11010 (Molecular Probes, Invitrogen detection technologies, Eugene, OR.

    Article Title: Novel peptide probes to assess the tensional state of fibronectin fibers in cancer
    Article Snippet: Paragraph title: Preparation and staining of histological tissue sections ... Primary antibody solution was removed and samples were washed before incubation with secondary goat anti-rabbit Alexa-633 (A-21070, Invitrogen, 1:100 dilution) or goat anti rat Alexa 546 (A-11081, Invitrogen, 1:100 dilution) or goat anti-rabbit Alexa-488 (A-11034, Invitrogen, 1:100 dilution) antibody solution for 45 min. After another washing step, samples were mounted using Prolong Antifade Gold with or without DAPI (Invitrogen).

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway
    Article Snippet: EC in artery sections were stained with mouse anti-human CD31 antibody (1:100; clone 89C2,Cell Signaling Technology,). .. Antibody binding was visualized with Alexa Fluor 594 anti-mouse IgG (1:200; A-11032, Thermo Fisher Scientific) and Alexa Fluor 488 anti-rabbit IgG (1:200; A-11034, Thermo Fisher Scientific) as secondary antibodies, or developed with the VECTASTAIN ABC kit (Vector Laboratories) plus DAB (3,3´-diaminobenzidine) substrate (Vector Laboratories).

    Article Title: Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137
    Article Snippet: .. Secondary antibodies for immunofluorescence staining include Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Invitrogen, A-11034) and Alexa Fluor® 546-conjugated goat anti-mouse IgG (Invitrogen, A-11030). .. Agarose conjugated with secondary anti-mouse IgG (A6531) and EZview Red affinity gel conjugated with rabbit anti-c-Myc antibody (E6654) were purchased from Sigma.

    Article Title: Elevation of pro-inflammatory cytokine levels following anti-resorptive drug treatment is required for osteonecrosis development in infectious osteomyelitis
    Article Snippet: .. Immunofluorescence Surgical sections of bone tissue were stained using a MEBSTAIN Apoptosis TUNEL Kit Direct (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan), or stained with anti-single standard DNA (ssDNA) Rabbit IgG (#18731 1:50; IBL, Gunma, Japan) followed by Alexa488-conjugated goat anti-rabbit Ig’ (#A-11034 1:200; Invitrogen, Carlsbad, CA). .. Sections were also stained with Alexa Fluor 488-conjugated rat anti-mouse F4/80 (#B116640 1:100 BioLegend, San Diego, CA, USA) and goat anti-mouse TNFα (#K2911 1:100 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by Alexa488-conjugated donkey anti-rat Ig’ (#A-21208 1:200; Invitrogen, Carlsbad, CA) and Alexa568-conjugated donkey anti-goat Ig’ (#A-11057 1:200; Invitrogen, Carlsbad, CA).

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  • 90
    Thermo Fisher af647 conjugated secondary antibodies
    Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with <t>AF647</t> (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).
    Af647 Conjugated Secondary Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 21 article reviews
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    af647 conjugated secondary antibodies - by Bioz Stars, 2020-02
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    90
    Thermo Fisher goat anti rabbit igg3
    USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control <t>IgG</t> or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.
    Goat Anti Rabbit Igg3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit anti goat igg alexa fluor 594
    Surface expression comparison of B16-F1-derived vesicles using flow cytometry ( A ) Vesicles conjugated to aldehyde-sulfate microspheres were analyzed by flow cytometry using biotinylated (bio) antibodies for the indicated tetraspanins, adhesion molecules, and clotting factors; bio-annexin V for phosphatidylserine (PS) and goat anti-mouse tissue factor (TF). ( B ) TF expression on B16-F1 cells, EL4, or TF-transfected EL4 (EL4-TF) as analyzed by flow cytometry. Soluble TF (sTF) was used to neutralise the anti-TF polyclonal antibody. Biotin was detected using streptavidin-allophycocyanin (SA-APC), and TF was detected using rabbit anti-goat <t>IgG</t> <t>Alexa</t> Fluor ® 594. Grey shaded peaks represent BSA-bead control, goat IgG control for TF bead samples; black lines represent EV-beads or cells; dotted lines represent TF antibody neutralized cells. ( C ) Vesicle lysates were subjected to PAGE and Western blotted with goat anti-mouse CD147 (detected with anti-goat IgG-horse radish peroxidase (HRP), mouse anti-mouse clathrin heavy chain, and mouse anti-mouse β actin IgG-HRP (detected with anti-mouse IgG-HRP). MW in kDa are shown. Results are representative of at least two experiments. ( D ) TOP3 precursor ion intensities [ 69 ] normalised to β-actin (y-axis) are represented in rank order (x-axis) in the exosome proteome for the three vesicle types.
    Rabbit Anti Goat Igg Alexa Fluor 594, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).

    Journal: Journal of Virology

    Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

    doi: 10.1128/JVI.00537-17

    Figure Lengend Snippet: Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).

    Article Snippet: Primary antibodies were detected using pAb goat anti-rabbit or anti-mouse AF488 or AF647 conjugated secondary antibodies (A-11034, A-11029, A-21244, and A-21236; Thermo Fisher Scientific).

    Techniques: Infection, Live Cell Imaging, Labeling, Incubation, Western Blot

    L1 proteins that accompany the viral genome into the nucleus dissociate after release of the viral genome. (A) At 24 h, uninfected HaCaT cells were fixed, permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100, and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were incubated with pAb rabbit anti-TGN46 (cyan), which recognizes a luminal epitope of TGN46. Lastly, the cells were permeabilized in 0.5% TX-100, followed by incubation with goat anti-rabbit secondary antibody and subsequent mounting in DAPI (white). Note the lack of reactivity of luminal anti-TGN46 antibody after digitonin treatment. (B and C) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml (B) or 0.5% TX-100 (C), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L1-7 (cyan) for specific detection of the L1 protein and mounted in DAPI (white). (D and E) The percent accessibility of viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible (AC) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L1 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L1 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells, and > 800 EdU puncta were counted per experiment). Mitosis: %IN = 88.58% ± 7.67%, %AC = 11.42% ± 7.67%, %L1 of IN = 82.3% ± 7.30%, %L1 of AC = 42.665% ± 9.335%. Interphase: %IN = 34.53% ± 0.427%, %AC = 68.36% ± 6.463%, %L1 of IN = 58.8% ± 3.805%, %L1 of AC = 20.84% ± 9.159%.

    Journal: Journal of Virology

    Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

    doi: 10.1128/JVI.00537-17

    Figure Lengend Snippet: L1 proteins that accompany the viral genome into the nucleus dissociate after release of the viral genome. (A) At 24 h, uninfected HaCaT cells were fixed, permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100, and treated with AF555 (green) in Click-iT reaction buffer. Next, the cells were incubated with pAb rabbit anti-TGN46 (cyan), which recognizes a luminal epitope of TGN46. Lastly, the cells were permeabilized in 0.5% TX-100, followed by incubation with goat anti-rabbit secondary antibody and subsequent mounting in DAPI (white). Note the lack of reactivity of luminal anti-TGN46 antibody after digitonin treatment. (B and C) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml (B) or 0.5% TX-100 (C), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L1-7 (cyan) for specific detection of the L1 protein and mounted in DAPI (white). (D and E) The percent accessibility of viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible (AC) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L1 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L1 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells, and > 800 EdU puncta were counted per experiment). Mitosis: %IN = 88.58% ± 7.67%, %AC = 11.42% ± 7.67%, %L1 of IN = 82.3% ± 7.30%, %L1 of AC = 42.665% ± 9.335%. Interphase: %IN = 34.53% ± 0.427%, %AC = 68.36% ± 6.463%, %L1 of IN = 58.8% ± 3.805%, %L1 of AC = 20.84% ± 9.159%.

    Article Snippet: Primary antibodies were detected using pAb goat anti-rabbit or anti-mouse AF488 or AF647 conjugated secondary antibodies (A-11034, A-11029, A-21244, and A-21236; Thermo Fisher Scientific).

    Techniques: Incubation, Infection, Labeling, Staining

    L2 proteins that accompany the viral genome into the nucleus remain associated with the viral genome. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml of (A) or 0.5% TX-100 (B), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L2-1 (cyan) for specific detection of the L2 protein and mounted in DAPI (white). (C and D) The percent accessibility of the viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible [AC]) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L2 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L2 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells and > 800 EdU puncta counter per experiment). Mitosis: %IN = 83.67% ± 8.435%, %AC = 16.34% ± 8.435%, %L2 of IN = 46.15% ± 6.15%, %L2 of AC = 24.45% ± 16.55%. Interphase: %IN = 42.15% ± 1.35%, %AC = 57.85% ± 1.35%, %L2 of IN = 44.65% ± 6.35%, %L2 of AC = 30.65% ± 3.35%.

    Journal: Journal of Virology

    Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

    doi: 10.1128/JVI.00537-17

    Figure Lengend Snippet: L2 proteins that accompany the viral genome into the nucleus remain associated with the viral genome. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized with digitonin at 0.625 μg/ml of (A) or 0.5% TX-100 (B), and treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were incubated with mouse MAb 33L2-1 (cyan) for specific detection of the L2 protein and mounted in DAPI (white). (C and D) The percent accessibility of the viral genome was determined by counting the number of red-only (inaccessible [IN]) or red/green (accessible [AC]) stained EdU puncta associated with condensed chromosomes on mitotic cells or nuclear localized in interphase cells. Colocalization of L2 and EdU puncta was quantified by counting the number of EdU puncta that colocalized with L2 signal. Quantifications are from two repeat experiments analyzing two to three Z-stack images per cell ( n = 30 to 40 cells and > 800 EdU puncta counter per experiment). Mitosis: %IN = 83.67% ± 8.435%, %AC = 16.34% ± 8.435%, %L2 of IN = 46.15% ± 6.15%, %L2 of AC = 24.45% ± 16.55%. Interphase: %IN = 42.15% ± 1.35%, %AC = 57.85% ± 1.35%, %L2 of IN = 44.65% ± 6.35%, %L2 of AC = 30.65% ± 3.35%.

    Article Snippet: Primary antibodies were detected using pAb goat anti-rabbit or anti-mouse AF488 or AF647 conjugated secondary antibodies (A-11034, A-11029, A-21244, and A-21236; Thermo Fisher Scientific).

    Techniques: Infection, Labeling, Incubation, Staining

    Adventitial mvECs in GCA arteries express Jagged1 Tissue biopsies were collected from temporal arteries and from aortic wall specimens. Arteries affected by GCA had typical transmural granulomatous arteritis (GCA-positive and GCA aortitis). Temporal arteries with no inflammatory infiltrates (GCA-negative) served as controls. Nuclei were marked with 4´,6-diamidino-2-phenylindole. Scale bars 50 μm. (A) Tissue-infiltrating T cells were identified by staining sections from GCA-negative and GCA-positive temporal arteries and from GCA aortitis with mouse anti-human CD3 antibody. Antibody binding was visualized with Alexa Fluor 594-labeled anti-mouse immunoglobulin G (IgG) secondary antibody (red). Representative stains from eight samples each are shown here. (B) mRNA was extracted from GCA-positive and GCA-negative temporal arteries and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the expression of JAG1 , DLL1 and DLL4 transcripts. Results (mean ± SEM) from six GCA arteries and six healthy arteries are shown. N.s., not significant. (C and D) Dual-color staining was applied to identify Jagged1 and Delta-like 1 expressed on endothelial cells in GCA-positive, GCA aortitis, and GCA-negative arteries. Tissue sections were double-stained with mouse anti-human CD31 antibody and rabbit anti-human Jagged1 antibody or rabbit anti-human Delta-like 1 antibody. Alexa Fluor 594 anti-mouse IgG (red) and Alexa Fluor 488 anti-rabbit IgG (green) were used as secondary antibodies. Merged images demonstrate colocalization of both markers (yellow). Representative images from eight samples each.

    Journal: Science translational medicine

    Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway

    doi: 10.1126/scitranslmed.aal3322

    Figure Lengend Snippet: Adventitial mvECs in GCA arteries express Jagged1 Tissue biopsies were collected from temporal arteries and from aortic wall specimens. Arteries affected by GCA had typical transmural granulomatous arteritis (GCA-positive and GCA aortitis). Temporal arteries with no inflammatory infiltrates (GCA-negative) served as controls. Nuclei were marked with 4´,6-diamidino-2-phenylindole. Scale bars 50 μm. (A) Tissue-infiltrating T cells were identified by staining sections from GCA-negative and GCA-positive temporal arteries and from GCA aortitis with mouse anti-human CD3 antibody. Antibody binding was visualized with Alexa Fluor 594-labeled anti-mouse immunoglobulin G (IgG) secondary antibody (red). Representative stains from eight samples each are shown here. (B) mRNA was extracted from GCA-positive and GCA-negative temporal arteries and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the expression of JAG1 , DLL1 and DLL4 transcripts. Results (mean ± SEM) from six GCA arteries and six healthy arteries are shown. N.s., not significant. (C and D) Dual-color staining was applied to identify Jagged1 and Delta-like 1 expressed on endothelial cells in GCA-positive, GCA aortitis, and GCA-negative arteries. Tissue sections were double-stained with mouse anti-human CD31 antibody and rabbit anti-human Jagged1 antibody or rabbit anti-human Delta-like 1 antibody. Alexa Fluor 594 anti-mouse IgG (red) and Alexa Fluor 488 anti-rabbit IgG (green) were used as secondary antibodies. Merged images demonstrate colocalization of both markers (yellow). Representative images from eight samples each.

    Article Snippet: Antibody binding was visualized with Alexa Fluor 594 anti-mouse IgG (1:200; A-11032, Thermo Fisher Scientific) and Alexa Fluor 488 anti-rabbit IgG (1:200; A-11034, Thermo Fisher Scientific) as secondary antibodies, or developed with the VECTASTAIN ABC kit (Vector Laboratories) plus DAB (3,3´-diaminobenzidine) substrate (Vector Laboratories).

    Techniques: Staining, Binding Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing

    USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.

    Journal: The Journal of Biological Chemistry

    Article Title: Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1)

    doi: 10.1074/jbc.RA118.003616

    Figure Lengend Snippet: USF-1 interacts with the ILEI promoter sequence. A, 5′-biotin–tagged ILEI promoter oligonucleotide constructs with WT or mutant E-box. Biotin pulldown analysis of nuclear extracts from 501-Mel melanoma cells, followed by immunoblot for USF-1. B, PCR primers flanking the ILEI promoter E-box used in ChIP analysis. ChIP analysis of 501-Mel melanoma cell lines immunoprecipitated with control IgG or USF-1 antibody. PCR analysis conducted with primers targeting FAM3C, TYR, or HO1 promoter.

    Article Snippet: The following secondary antibodies were used: goat anti-mouse IgG (31430; ThermoFisher; 1:10,000) and goat anti-rabbit IgG3 (31460; ThermoFisher; 1:10,000).

    Techniques: Sequencing, Construct, Mutagenesis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

    Surface expression comparison of B16-F1-derived vesicles using flow cytometry ( A ) Vesicles conjugated to aldehyde-sulfate microspheres were analyzed by flow cytometry using biotinylated (bio) antibodies for the indicated tetraspanins, adhesion molecules, and clotting factors; bio-annexin V for phosphatidylserine (PS) and goat anti-mouse tissue factor (TF). ( B ) TF expression on B16-F1 cells, EL4, or TF-transfected EL4 (EL4-TF) as analyzed by flow cytometry. Soluble TF (sTF) was used to neutralise the anti-TF polyclonal antibody. Biotin was detected using streptavidin-allophycocyanin (SA-APC), and TF was detected using rabbit anti-goat IgG Alexa Fluor ® 594. Grey shaded peaks represent BSA-bead control, goat IgG control for TF bead samples; black lines represent EV-beads or cells; dotted lines represent TF antibody neutralized cells. ( C ) Vesicle lysates were subjected to PAGE and Western blotted with goat anti-mouse CD147 (detected with anti-goat IgG-horse radish peroxidase (HRP), mouse anti-mouse clathrin heavy chain, and mouse anti-mouse β actin IgG-HRP (detected with anti-mouse IgG-HRP). MW in kDa are shown. Results are representative of at least two experiments. ( D ) TOP3 precursor ion intensities [ 69 ] normalised to β-actin (y-axis) are represented in rank order (x-axis) in the exosome proteome for the three vesicle types.

    Journal: Oncotarget

    Article Title: Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles

    doi: 10.18632/oncotarget.10783

    Figure Lengend Snippet: Surface expression comparison of B16-F1-derived vesicles using flow cytometry ( A ) Vesicles conjugated to aldehyde-sulfate microspheres were analyzed by flow cytometry using biotinylated (bio) antibodies for the indicated tetraspanins, adhesion molecules, and clotting factors; bio-annexin V for phosphatidylserine (PS) and goat anti-mouse tissue factor (TF). ( B ) TF expression on B16-F1 cells, EL4, or TF-transfected EL4 (EL4-TF) as analyzed by flow cytometry. Soluble TF (sTF) was used to neutralise the anti-TF polyclonal antibody. Biotin was detected using streptavidin-allophycocyanin (SA-APC), and TF was detected using rabbit anti-goat IgG Alexa Fluor ® 594. Grey shaded peaks represent BSA-bead control, goat IgG control for TF bead samples; black lines represent EV-beads or cells; dotted lines represent TF antibody neutralized cells. ( C ) Vesicle lysates were subjected to PAGE and Western blotted with goat anti-mouse CD147 (detected with anti-goat IgG-horse radish peroxidase (HRP), mouse anti-mouse clathrin heavy chain, and mouse anti-mouse β actin IgG-HRP (detected with anti-mouse IgG-HRP). MW in kDa are shown. Results are representative of at least two experiments. ( D ) TOP3 precursor ion intensities [ 69 ] normalised to β-actin (y-axis) are represented in rank order (x-axis) in the exosome proteome for the three vesicle types.

    Article Snippet: Following washing with 0.05% BSA/PBS, biotin was detected using 1 μg/mL APC-conjugated streptavidin (BioLegend #405207), primary anti-TF antibody was detected with 2 μg/mL rabbit anti-goat IgG Alexa Fluor® 594 (ThermoFisher #A-11080).

    Techniques: Expressing, Derivative Assay, Flow Cytometry, Cytometry, Coagulation, Transfection, Polyacrylamide Gel Electrophoresis, Western Blot