ecorv hf  (New England Biolabs)


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    New England Biolabs ecorv hf
    SeqStain-based multiplex immunofluorescence imaging (A) SeqStain methodology schematic. Immobilized cells and tissue sections are processed in multiple, sequential cycles of immunostaining with fluorescent DNA-labeled antibodies, imaging, gentle de-staining using a nuclease, and re-imaging. Post imaging, the data are analyzed by computational stacking and alignment of the images to generate spatial relationship maps. The schematic was generated using Biorender. (B) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either <t>DNase</t> I (top panels) or the endonuclease <t>EcoRV</t> (bottom panels). All images are representative of at least three replicates. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. (C) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labeled with AF488 fluorophore) or anti-CD45 (labeled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labeled using DAPI. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (D) Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 min after the addition of DNase I (bottom panel). All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (E) Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labeled using the AF488 fluorophore shown in green and the antibodies labeled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is presented on the right. Graphs show the mean ± standard deviation (SD). Scale bars, 100 μm.
    Ecorv Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "SeqStain is an efficient method for multiplexed, spatialomic profiling of human and murine tissues"

    Article Title: SeqStain is an efficient method for multiplexed, spatialomic profiling of human and murine tissues

    Journal: Cell reports methods

    doi: 10.1016/j.crmeth.2021.100006

    SeqStain-based multiplex immunofluorescence imaging (A) SeqStain methodology schematic. Immobilized cells and tissue sections are processed in multiple, sequential cycles of immunostaining with fluorescent DNA-labeled antibodies, imaging, gentle de-staining using a nuclease, and re-imaging. Post imaging, the data are analyzed by computational stacking and alignment of the images to generate spatial relationship maps. The schematic was generated using Biorender. (B) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. (C) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labeled with AF488 fluorophore) or anti-CD45 (labeled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labeled using DAPI. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (D) Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 min after the addition of DNase I (bottom panel). All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (E) Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labeled using the AF488 fluorophore shown in green and the antibodies labeled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is presented on the right. Graphs show the mean ± standard deviation (SD). Scale bars, 100 μm.
    Figure Legend Snippet: SeqStain-based multiplex immunofluorescence imaging (A) SeqStain methodology schematic. Immobilized cells and tissue sections are processed in multiple, sequential cycles of immunostaining with fluorescent DNA-labeled antibodies, imaging, gentle de-staining using a nuclease, and re-imaging. Post imaging, the data are analyzed by computational stacking and alignment of the images to generate spatial relationship maps. The schematic was generated using Biorender. (B) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. (C) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labeled with AF488 fluorophore) or anti-CD45 (labeled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labeled using DAPI. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (D) Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 min after the addition of DNase I (bottom panel). All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (E) Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labeled using the AF488 fluorophore shown in green and the antibodies labeled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is presented on the right. Graphs show the mean ± standard deviation (SD). Scale bars, 100 μm.

    Techniques Used: Multiplex Assay, Immunofluorescence, Imaging, Immunostaining, Labeling, Staining, Generated, Fluorescence, Standard Deviation

    2) Product Images from "The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II"

    Article Title: The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2021.100734

    CKM is excluded from cMed–PIC and competes with pol II for cMed binding. A , to investigate whether the CKM binds to the PIC together with cMed, core PIC components were added as well as cMed and CKM to biotinylated promoter DNA immobilized on streptavidin beads with a downstream EcoRV restriction digestion site, to allow specific elution. CKM(A) alone, cMed alone, and CKM(A)–cMed alone had only minute background binding to promoter DNA (lanes 1, 2, and 3). Pol II, initiation factors, and cMed bound to promoter DNA forming a cMed–PIC complex (lane 4). CKM did not bind to cMed–PIC above background levels, and this was irrespective of whether CKM(A) (lane 5), CKM(A) in the presence of ATP (lane 6), or CKM(KD) in the presence of ATP (lane 7) was used, indicating that the exclusion of the CKM from cMed–PIC is a steric structural effect, rather than a kinase-dependent one. The unbound components corresponding to each lane (1–7) are shown in the same order in lanes 8 to 14. Characteristic complex bands are indicated with rectangles of the appropriate color for clarity. B , CKM(KD) immobilized on amylose beads was saturated with an excess of cMed ( top ) and then washed with increasing concentrations of pol II ( left to right ), and the washes were probed with an anti-Med17 (a cMed subunit) antibody. Pol II competed cMed away from CKM binding as indicated by an increase in the anti-Med17 signal in the washes with increasing added pol II. The same experiment but with no added cMed ( bottom ) showed no change in the anti-Med17 signal with increasing added pol II. CKM, Cdk8 kinase module; cMed, core mediator; PIC, preinitiation complex; pol II, RNA polymerase II.
    Figure Legend Snippet: CKM is excluded from cMed–PIC and competes with pol II for cMed binding. A , to investigate whether the CKM binds to the PIC together with cMed, core PIC components were added as well as cMed and CKM to biotinylated promoter DNA immobilized on streptavidin beads with a downstream EcoRV restriction digestion site, to allow specific elution. CKM(A) alone, cMed alone, and CKM(A)–cMed alone had only minute background binding to promoter DNA (lanes 1, 2, and 3). Pol II, initiation factors, and cMed bound to promoter DNA forming a cMed–PIC complex (lane 4). CKM did not bind to cMed–PIC above background levels, and this was irrespective of whether CKM(A) (lane 5), CKM(A) in the presence of ATP (lane 6), or CKM(KD) in the presence of ATP (lane 7) was used, indicating that the exclusion of the CKM from cMed–PIC is a steric structural effect, rather than a kinase-dependent one. The unbound components corresponding to each lane (1–7) are shown in the same order in lanes 8 to 14. Characteristic complex bands are indicated with rectangles of the appropriate color for clarity. B , CKM(KD) immobilized on amylose beads was saturated with an excess of cMed ( top ) and then washed with increasing concentrations of pol II ( left to right ), and the washes were probed with an anti-Med17 (a cMed subunit) antibody. Pol II competed cMed away from CKM binding as indicated by an increase in the anti-Med17 signal in the washes with increasing added pol II. The same experiment but with no added cMed ( bottom ) showed no change in the anti-Med17 signal with increasing added pol II. CKM, Cdk8 kinase module; cMed, core mediator; PIC, preinitiation complex; pol II, RNA polymerase II.

    Techniques Used: Binding Assay

    3) Product Images from "The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing"

    Article Title: The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00070

    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.
    Figure Legend Snippet: (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Techniques Used: Plasmid Preparation, Expressing, Immunocytochemistry, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Transfection, Isolation, Purification, Clone Assay

    4) Product Images from "SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues"

    Article Title: SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

    Journal: bioRxiv

    doi: 10.1101/2020.11.16.385237

    SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.
    Figure Legend Snippet: SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.

    Techniques Used: Multiplex Assay, Immunofluorescence, Imaging, Immunostaining, Staining, Generated, Fluorescence, Standard Deviation

    5) Product Images from "Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism"

    Article Title: Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism

    Journal: bioRxiv

    doi: 10.1101/2020.07.20.211623

    Schematics illustrating the cloning strategy. (A) The three parental vectors used for generating untagged, N-, and C-terminally tagged constructs. (B) Features common to all synthesized insert sequences. Each insert included BamHI and EcoRV sites at either end to facilitate cloning into the three parental vectors. To allow for C-terminal cloning, an AvrII site was inserted such that it overlapped the stop codon (see text for details). In order to accommodate the AvrII site, an alanine residue was added to the end of each expression construct. The viral ORFs were codon-optimized for moderate or high expression, and lacked BamHI, AvrII, and EcoRV sites. (C) For untagged and N-terminal tagging, inserts were digested with BamHI and EcoRV and ligated directly into plasmid precut with the same enzymes. For C-terminal cloning, the inserts were first digested with AvrII, blunted with Mung Bean Nuclease, and then cut with BamHI. The resulting fragment was ligated into plasmid cut with BamHI and EcoRV. (D) Untagged, N-, and C-terminally tagged expression constructs. (E) Strategy for generating stable cell lines (see text for details).
    Figure Legend Snippet: Schematics illustrating the cloning strategy. (A) The three parental vectors used for generating untagged, N-, and C-terminally tagged constructs. (B) Features common to all synthesized insert sequences. Each insert included BamHI and EcoRV sites at either end to facilitate cloning into the three parental vectors. To allow for C-terminal cloning, an AvrII site was inserted such that it overlapped the stop codon (see text for details). In order to accommodate the AvrII site, an alanine residue was added to the end of each expression construct. The viral ORFs were codon-optimized for moderate or high expression, and lacked BamHI, AvrII, and EcoRV sites. (C) For untagged and N-terminal tagging, inserts were digested with BamHI and EcoRV and ligated directly into plasmid precut with the same enzymes. For C-terminal cloning, the inserts were first digested with AvrII, blunted with Mung Bean Nuclease, and then cut with BamHI. The resulting fragment was ligated into plasmid cut with BamHI and EcoRV. (D) Untagged, N-, and C-terminally tagged expression constructs. (E) Strategy for generating stable cell lines (see text for details).

    Techniques Used: Clone Assay, Construct, Synthesized, Expressing, Plasmid Preparation, Stable Transfection

    6) Product Images from "Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions"

    Article Title: Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

    Journal: bioRxiv

    doi: 10.1101/203919

    Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).
    Figure Legend Snippet: Complexity Reduction Via Restriction Enzyme Degradation Increases the Number of On-Target Reads Number of CCS reads (individual molecules) is plotted for each of the four target regions ( C9orf72 , HTT , FMR1 , and ATXN10 ). Colors represent the amount of complexity reduction employed (blue = none; orange = two restriction enzymes (KpnI-HF and MfeI-HF), green = four restriction enzymes (KpnI-HF, Mfe-HFI, SpeI-HF, EcoRV-HF)).

    Techniques Used:

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    New England Biolabs ecorv hf
    SeqStain-based multiplex immunofluorescence imaging (A) SeqStain methodology schematic. Immobilized cells and tissue sections are processed in multiple, sequential cycles of immunostaining with fluorescent DNA-labeled antibodies, imaging, gentle de-staining using a nuclease, and re-imaging. Post imaging, the data are analyzed by computational stacking and alignment of the images to generate spatial relationship maps. The schematic was generated using Biorender. (B) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either <t>DNase</t> I (top panels) or the endonuclease <t>EcoRV</t> (bottom panels). All images are representative of at least three replicates. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. (C) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labeled with AF488 fluorophore) or anti-CD45 (labeled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labeled using DAPI. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (D) Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 min after the addition of DNase I (bottom panel). All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (E) Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labeled using the AF488 fluorophore shown in green and the antibodies labeled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is presented on the right. Graphs show the mean ± standard deviation (SD). Scale bars, 100 μm.
    Ecorv Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecorv hf/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    SeqStain-based multiplex immunofluorescence imaging (A) SeqStain methodology schematic. Immobilized cells and tissue sections are processed in multiple, sequential cycles of immunostaining with fluorescent DNA-labeled antibodies, imaging, gentle de-staining using a nuclease, and re-imaging. Post imaging, the data are analyzed by computational stacking and alignment of the images to generate spatial relationship maps. The schematic was generated using Biorender. (B) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. (C) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labeled with AF488 fluorophore) or anti-CD45 (labeled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labeled using DAPI. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (D) Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 min after the addition of DNase I (bottom panel). All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (E) Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labeled using the AF488 fluorophore shown in green and the antibodies labeled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is presented on the right. Graphs show the mean ± standard deviation (SD). Scale bars, 100 μm.

    Journal: Cell reports methods

    Article Title: SeqStain is an efficient method for multiplexed, spatialomic profiling of human and murine tissues

    doi: 10.1016/j.crmeth.2021.100006

    Figure Lengend Snippet: SeqStain-based multiplex immunofluorescence imaging (A) SeqStain methodology schematic. Immobilized cells and tissue sections are processed in multiple, sequential cycles of immunostaining with fluorescent DNA-labeled antibodies, imaging, gentle de-staining using a nuclease, and re-imaging. Post imaging, the data are analyzed by computational stacking and alignment of the images to generate spatial relationship maps. The schematic was generated using Biorender. (B) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. (C) Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labeled with AF488 fluorophore) or anti-CD45 (labeled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labeled using DAPI. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (D) Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 min after the addition of DNase I (bottom panel). All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. (E) Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labeled using the AF488 fluorophore shown in green and the antibodies labeled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is presented on the right. Graphs show the mean ± standard deviation (SD). Scale bars, 100 μm.

    Article Snippet: Block-1 solution (PBS containing 1% Bovine Serum Albumin (Sigma-Aldrich, #B4287–1G)), Block-2 solution (PBS containing 200ng/ml salmon sperm DNA (Thermo Fisher, #AM9680) and 3 nano-moles/ml single-stranded DNA and 0.5M NaCl), Wash buffer (PBS containing 0.1% Tween-20 (Sigma-Aldrich, #P1379), DNase I de-staining buffer (PBS containing 1X DNase buffer and 20 units of DNase I (NEB, #M0303) in 500ul), EcoRV de-staining buffer (de-ionized water containing 1X cut-smart buffer and 200 units of EcoRV (NEB, #R3195S) in 500ul), SmaI de-staining buffer (de-ionized water containing 1X cut smart buffer and 200 units of SmaI (NEB, #R01041S) in 500ul).

    Techniques: Multiplex Assay, Immunofluorescence, Imaging, Immunostaining, Labeling, Staining, Generated, Fluorescence, Standard Deviation

    CKM is excluded from cMed–PIC and competes with pol II for cMed binding. A , to investigate whether the CKM binds to the PIC together with cMed, core PIC components were added as well as cMed and CKM to biotinylated promoter DNA immobilized on streptavidin beads with a downstream EcoRV restriction digestion site, to allow specific elution. CKM(A) alone, cMed alone, and CKM(A)–cMed alone had only minute background binding to promoter DNA (lanes 1, 2, and 3). Pol II, initiation factors, and cMed bound to promoter DNA forming a cMed–PIC complex (lane 4). CKM did not bind to cMed–PIC above background levels, and this was irrespective of whether CKM(A) (lane 5), CKM(A) in the presence of ATP (lane 6), or CKM(KD) in the presence of ATP (lane 7) was used, indicating that the exclusion of the CKM from cMed–PIC is a steric structural effect, rather than a kinase-dependent one. The unbound components corresponding to each lane (1–7) are shown in the same order in lanes 8 to 14. Characteristic complex bands are indicated with rectangles of the appropriate color for clarity. B , CKM(KD) immobilized on amylose beads was saturated with an excess of cMed ( top ) and then washed with increasing concentrations of pol II ( left to right ), and the washes were probed with an anti-Med17 (a cMed subunit) antibody. Pol II competed cMed away from CKM binding as indicated by an increase in the anti-Med17 signal in the washes with increasing added pol II. The same experiment but with no added cMed ( bottom ) showed no change in the anti-Med17 signal with increasing added pol II. CKM, Cdk8 kinase module; cMed, core mediator; PIC, preinitiation complex; pol II, RNA polymerase II.

    Journal: The Journal of Biological Chemistry

    Article Title: The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II

    doi: 10.1016/j.jbc.2021.100734

    Figure Lengend Snippet: CKM is excluded from cMed–PIC and competes with pol II for cMed binding. A , to investigate whether the CKM binds to the PIC together with cMed, core PIC components were added as well as cMed and CKM to biotinylated promoter DNA immobilized on streptavidin beads with a downstream EcoRV restriction digestion site, to allow specific elution. CKM(A) alone, cMed alone, and CKM(A)–cMed alone had only minute background binding to promoter DNA (lanes 1, 2, and 3). Pol II, initiation factors, and cMed bound to promoter DNA forming a cMed–PIC complex (lane 4). CKM did not bind to cMed–PIC above background levels, and this was irrespective of whether CKM(A) (lane 5), CKM(A) in the presence of ATP (lane 6), or CKM(KD) in the presence of ATP (lane 7) was used, indicating that the exclusion of the CKM from cMed–PIC is a steric structural effect, rather than a kinase-dependent one. The unbound components corresponding to each lane (1–7) are shown in the same order in lanes 8 to 14. Characteristic complex bands are indicated with rectangles of the appropriate color for clarity. B , CKM(KD) immobilized on amylose beads was saturated with an excess of cMed ( top ) and then washed with increasing concentrations of pol II ( left to right ), and the washes were probed with an anti-Med17 (a cMed subunit) antibody. Pol II competed cMed away from CKM binding as indicated by an increase in the anti-Med17 signal in the washes with increasing added pol II. The same experiment but with no added cMed ( bottom ) showed no change in the anti-Med17 signal with increasing added pol II. CKM, Cdk8 kinase module; cMed, core mediator; PIC, preinitiation complex; pol II, RNA polymerase II.

    Article Snippet: About 24.5 μl of streptavidin-binding buffer + 0.5 μl of 100 U/μl EcoRV-HF (New England Biolabs) were added per tube and incubated at 28 °C for 75 min to allow the restriction enzyme to cleave off the DNA from the beads.

    Techniques: Binding Assay

    (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    doi: 10.3389/fnmol.2016.00070

    Figure Lengend Snippet: (A) AAV vector maps depicting AAV-P Tight -Cas9 and AAV-gRNA/rtTA. AAV-P Tight -Cas9 consists of a Cas9 transgene under the control of a Dox inducible Tight promoter. AAV-gRNA/rtTA consists of a gRNA expression cassette and a rtTA (Tet-On Advanced) transgene controlled by a CMV promoter. It also is designed to express GFP via an IRES element following the rtTA reading frame. (B) ICC for Cas9 and GFP was performed on 293FT cells transduced by AAV-P Tight -Cas9 and AAV-gRNA/rtTA viruses in the presence or absence of Dox. Native GFP expression is visible in virtually all of the cells (i, iii). Cas9 expression is robustly induced in the presence of Dox (ii), compared to the no Dox condition (iv). Representative images are shown. The experiment was repeated twice with similar results. (C) Diagram depicting the approximate location of where the Tet2 gRNA targets the Tet2 locus. Underlined nucleotides indicate the sequence of the Tet2 gRNA. Location of the EcoRV site and PAM sequence are denoted. (D) An approximate 460 bps region of the Tet2 locus that includes the site targeted for editing via the gRNA Tet2 , was PCR amplified from N2A genomic DNA and electrophoresed on a standard agarose gel and stained with ethidium bromide (lane 1). N2A cells were transfected with the pX330 Empty , a plasmid designed to express spCas9 and no gRNA, and 96 h later, the genomic DNA was isolated and the Tet2 locus was PCR amplified and subjected to EcoRV digestion. The PCR product was cut into two pieces of DNA as expected (lane 2). However, when N2A cells were transfected with pX330 Tet2 and similarly processed, the PCR product was incompletely digested resulting in a total of three bands on the gel - one uncut PCR product (~460 bps) and two smaller bands. In this case the genome editing was ~33%. (E) Edited DNA depicted in ( D , lane 3) was gel purified and TA cloned and 6 independent clones were sequenced. These 6 clones contained deletions which destroyed the EcoRV site.

    Article Snippet: Three microliters of PCR product was digested with 5 units of EcoRV-HF (New England Biolabs) for 2 h in a standard 10 μl restriction enzyme reaction following the manufacturer's instructions.

    Techniques: Plasmid Preparation, Expressing, Immunocytochemistry, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Staining, Transfection, Isolation, Purification, Clone Assay

    SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.

    Journal: bioRxiv

    Article Title: SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

    doi: 10.1101/2020.11.16.385237

    Figure Lengend Snippet: SeqStain based multiplex immunofluorescence imaging. A . Schematic representing the SeqStain methodology. Immobilized cells and tissue sections on glass are processed in cycles of immuno-staining with fluorescent-DNA labelled antibodies (step 1), imaging (step 2), gentle destaining using a nuclease (step 3), re-imaging (step 4) followed by next round of staining steps (step 5). Post-imaging, the data is analysed by computational stacking and alignment of the images followed by analyses of the spatial relationships between various markers to generate spatial maps of molecules and cells. Schematics were generated using Biorender. B . Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 SeqStain antibody and after de-staining with either DNase I (top panels) or the endonuclease EcoRV (bottom panels). All images are representative of at least three replicates. Scale bar is 100μm. A bar graph showing quantification of fluorescence intensity for each panel is presented on the right. Graphs show the mean ± standard deviation. C. Immunofluorescence images of RAW264.7 cells after staining with anti-CD44 (fluorescently labelled with AF488 fluorophore) or anti-CD45 (labelled with Cy3 fluorophore) SeqStain antibodies (top panels) and after de-staining with DNase I for 1 min (bottom panels). Nuclei were labelled using DAPI. All images are representative of at least three replicates. Scale bar is 100 μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. D. Immunofluorescent images of RAW264.7 cells co-stained with anti-CD44 and anti-CD45 SeqStain antibodies (top panel) and 1 minute after the addition of DNase I (bottom panel). All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity in each panel is presented on the bottom. Graphs show the mean ± standard deviation. E. Immunofluorescence images of RAW264.7 cells after each of the three cycles of staining with two unique SeqStain antibodies and de-staining with DNase I. The antibodies used in each round are indicated in the panel, with SeqStain antibodies labelled using the AF488 fluorophore shown in green and the antibodies labelled using the Cy3 fluorophore shown in red. All images are representative of at least three replicates. Scale bar is 100μm. A graph showing quantification of fluorescence intensity after staining (green and red bars) and de-staining (brown bars) in each panel is also presented. Graphs show the mean ± standard deviation.

    Article Snippet: Block-1 solution (PBS containing 1% Bovine Serum Albumin (Sigma-Aldrich, #B4287-1G)), Block-2 solution (PBS containing 200ng/ml salmon sperm DNA (Thermo Fisher, #AM9680) and 3 nanomoles/ml singlestranded DNA and 0.5M NaCl), Wash buffer (PBS containing 0.1% Tween-20 (Sigma-Aldrich, #P1379), DNase I de-staining buffer (PBS containing 1X DNase buffer and 20 units of DNase I (NEB, #M0303) in 500ul), EcoRV de-staining buffer (deionized water containing 1X cut-smart buffer and 200 units of EcoRV (NEB, #R3195S) in 500ul), SmaI de-staining buffer (de-ionized water containing 1X cut smart buffer and 200 units of SmaI (NEB, #R01041S) in 500ul).

    Techniques: Multiplex Assay, Immunofluorescence, Imaging, Immunostaining, Staining, Generated, Fluorescence, Standard Deviation