vectabond  (Vector Laboratories)


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    Name:
    VECTABOND Reagent for Tissue Section Adhesion
    Description:
    VECTABOND Reagent is designed to significantly increase the adherence of both frozen and paraffin embedded tissue sections and cell preparations to glass slides and coverslips Tissue sections will remain attached even when subjected to the most extreme conditions such as high temperature antigen unmasking techniques and in situ hybridization VECTABOND Reagent treated slides can be stored for long periods This product chemically modifies the glass to form a highly adherent surface VECTABOND Reagent is provided as 7 ml of concentrate that dilutes to 350 ml of treatment solution sufficient for at least 500 standard slides
    Catalog Number:
    SP-1800
    Price:
    None
    Category:
    Nucleic acid non radioactive labeling kits
    Size:
    7 ml
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    Structured Review

    Vector Laboratories vectabond
    VECTABOND Reagent for Tissue Section Adhesion
    VECTABOND Reagent is designed to significantly increase the adherence of both frozen and paraffin embedded tissue sections and cell preparations to glass slides and coverslips Tissue sections will remain attached even when subjected to the most extreme conditions such as high temperature antigen unmasking techniques and in situ hybridization VECTABOND Reagent treated slides can be stored for long periods This product chemically modifies the glass to form a highly adherent surface VECTABOND Reagent is provided as 7 ml of concentrate that dilutes to 350 ml of treatment solution sufficient for at least 500 standard slides
    https://www.bioz.com/result/vectabond/product/Vector Laboratories
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vectabond - by Bioz Stars, 2021-06
    95/100 stars

    Images

    1) Product Images from "Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7"

    Article Title: Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008606

    The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope. (A) Schematic overview of trypsin shaving of E . coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E . coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm 2 scans of whole bacteria were shown on top and representative 500x500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm 2 scans and 2 deg for 500x500 nm 2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.
    Figure Legend Snippet: The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope. (A) Schematic overview of trypsin shaving of E . coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E . coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm 2 scans of whole bacteria were shown on top and representative 500x500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm 2 scans and 2 deg for 500x500 nm 2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.

    Techniques Used: Derivative Assay, Purification, Incubation, Flow Cytometry, Staining, Microscopy

    2) Product Images from "Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7"

    Article Title: Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.877639

    The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope A) Schematic overview of trypsin shaving of E. coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 µg/ml trypsin for 20 minutes ( open blue squares and open red circles ). Trypsin was inhibited with 50 µg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in ( A ) was plotted as geoMFI of the bacterial population. ( C ) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in ( A ). (D) Atomic force microscopy analysis (phase images) of E. coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in ( A ) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in ( A ). 2×2 µm 2 scans of whole bacteria were shown on top and representative 500×500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2×2 µm 2 scans and 2 deg for 500×500 nm 2 scans. Data represent with mean +-SD ( B and C ) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test ( B ). Significance was shown as * p ≤ 0.05.
    Figure Legend Snippet: The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope A) Schematic overview of trypsin shaving of E. coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 µg/ml trypsin for 20 minutes ( open blue squares and open red circles ). Trypsin was inhibited with 50 µg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in ( A ) was plotted as geoMFI of the bacterial population. ( C ) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in ( A ). (D) Atomic force microscopy analysis (phase images) of E. coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in ( A ) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in ( A ). 2×2 µm 2 scans of whole bacteria were shown on top and representative 500×500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2×2 µm 2 scans and 2 deg for 500×500 nm 2 scans. Data represent with mean +-SD ( B and C ) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test ( B ). Significance was shown as * p ≤ 0.05.

    Techniques Used: Derivative Assay, Purification, Incubation, Flow Cytometry, Staining, Microscopy

    3) Product Images from "Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7"

    Article Title: Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008606

    The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope. (A) Schematic overview of trypsin shaving of E . coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E . coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm 2 scans of whole bacteria were shown on top and representative 500x500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm 2 scans and 2 deg for 500x500 nm 2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.
    Figure Legend Snippet: The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope. (A) Schematic overview of trypsin shaving of E . coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E . coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm 2 scans of whole bacteria were shown on top and representative 500x500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm 2 scans and 2 deg for 500x500 nm 2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.

    Techniques Used: Derivative Assay, Purification, Incubation, Flow Cytometry, Staining, Microscopy

    Related Articles

    Immunohistochemistry:

    Article Title: Health Effects in Fish of Long-Term Exposure to Effluents from Wastewater Treatment Works
    Article Snippet: VTG immunohistochemistry We used immunohistochemistry to detect the presence and assess the distribution of VTG in the body tissues of 10 male and 10 female juvenile roach from the control and 80% effluent treatments, using a carp VTG polyclonal antibody (validated for use in the roach; ). .. For immunohistochemistry, glass microscope slides were treated with Vectabond (Vector Laboratories Ltd, Peterborough, UK) to aid slide attachment of paraffin sections and prevent detachment during processing. ..

    Microscopy:

    Article Title: Health Effects in Fish of Long-Term Exposure to Effluents from Wastewater Treatment Works
    Article Snippet: VTG immunohistochemistry We used immunohistochemistry to detect the presence and assess the distribution of VTG in the body tissues of 10 male and 10 female juvenile roach from the control and 80% effluent treatments, using a carp VTG polyclonal antibody (validated for use in the roach; ). .. For immunohistochemistry, glass microscope slides were treated with Vectabond (Vector Laboratories Ltd, Peterborough, UK) to aid slide attachment of paraffin sections and prevent detachment during processing. ..

    other:

    Article Title: Imaging live bacteria at the nanoscale: comparison of immobilisation strategies
    Article Snippet: Vectabond® Cleaned coverslips were put into a rack and submerged in 50 mL acetone for 5 minutes, then moved to a 50 : 1 solution of acetone : Vectabond® (Vector Laboratories, USA) for 5 minutes.

    Cell Culture:

    Article Title: Novel Modeling Approach to Generate a Polymeric Nanofiber Scaffold for Salivary Gland Cells
    Article Snippet: The exclusion of factors that failed the null hypothesis yielded the transfer functions which were further optimized using Minitab's optimizer tool and Microsoft Excel's solver tool. .. SIMS, an immortalized adult mouse submandibular salivary gland ductal epithelial cell line [ , ], was cultured in complete cell media, Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), and 100 U/ml penicillin and 100 μg/ml streptomycin, as previously described (all Invitrogen, Carlsbad, CA) PLGA fibers were electrospun onto 12 mm glass coverslips coated with Vectabond (Vector Laboratories) as per manufacturer's protocols and placed into individual wells in a 24-well tissue culture plate. ..

    Modification:

    Article Title: Novel Modeling Approach to Generate a Polymeric Nanofiber Scaffold for Salivary Gland Cells
    Article Snippet: The exclusion of factors that failed the null hypothesis yielded the transfer functions which were further optimized using Minitab's optimizer tool and Microsoft Excel's solver tool. .. SIMS, an immortalized adult mouse submandibular salivary gland ductal epithelial cell line [ , ], was cultured in complete cell media, Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), and 100 U/ml penicillin and 100 μg/ml streptomycin, as previously described (all Invitrogen, Carlsbad, CA) PLGA fibers were electrospun onto 12 mm glass coverslips coated with Vectabond (Vector Laboratories) as per manufacturer's protocols and placed into individual wells in a 24-well tissue culture plate. ..

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    Vector Laboratories vectabond
    The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope. (A) Schematic overview of trypsin shaving of E . coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E . coli MG1655 immobilized on <t>Vectabond</t> covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm 2 scans of whole bacteria were shown on top and representative 500x500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm 2 scans and 2 deg for 500x500 nm 2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.
    Vectabond, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vectabond/product/Vector Laboratories
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vectabond - by Bioz Stars, 2021-06
    95/100 stars
      Buy from Supplier

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    The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope. (A) Schematic overview of trypsin shaving of E . coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E . coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm 2 scans of whole bacteria were shown on top and representative 500x500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm 2 scans and 2 deg for 500x500 nm 2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.

    Journal: PLoS Pathogens

    Article Title: Bacterial killing by complement requires direct anchoring of membrane attack complex precursor C5b-7

    doi: 10.1371/journal.ppat.1008606

    Figure Lengend Snippet: The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope. (A) Schematic overview of trypsin shaving of E . coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 μg/ml trypsin for 20 minutes (open blue squares and open red circles). Trypsin was inhibited with 50 μg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in (A) was plotted as geoMFI of the bacterial population. (C) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in (A). (D) Atomic force microscopy analysis (phase images) of E . coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in (A) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in (A). 2x2 μm 2 scans of whole bacteria were shown on top and representative 500x500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2x2 μm 2 scans and 2 deg for 500x500 nm 2 scans. Data represent with mean +- SD (B and C) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test (B). Significance was shown as * p ≤ 0.05.

    Article Snippet: 100 μl of bacteria were immobilized on cleaned glass slides (Corning/Sigma Aldrich) covered with Vectabond® (Vector Laboratories, USA) as described previously for 30 minutes at RT [ ].

    Techniques: Derivative Assay, Purification, Incubation, Flow Cytometry, Staining, Microscopy

    The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope A) Schematic overview of trypsin shaving of E. coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 µg/ml trypsin for 20 minutes ( open blue squares and open red circles ). Trypsin was inhibited with 50 µg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in ( A ) was plotted as geoMFI of the bacterial population. ( C ) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in ( A ). (D) Atomic force microscopy analysis (phase images) of E. coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in ( A ) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in ( A ). 2×2 µm 2 scans of whole bacteria were shown on top and representative 500×500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2×2 µm 2 scans and 2 deg for 500×500 nm 2 scans. Data represent with mean +-SD ( B and C ) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test ( B ). Significance was shown as * p ≤ 0.05.

    Journal: bioRxiv

    Article Title: Bacterial killing by complement requires direct anchoring of Membrane Attack Complex precursor C5b-7

    doi: 10.1101/2019.12.17.877639

    Figure Lengend Snippet: The presence of C7 during C5b6 generation affects how C5b-7 is anchored to the bacterial cell envelope A) Schematic overview of trypsin shaving of E. coli MG1655 bacteria labelled with locally formed C5b-7 and C5b-7 derived from purified C5b6 (pC5b6). Convertase-labelled bacteria were incubated with 10 nM C5, 10 nM C6 and 10 nM C7 ( black squares ) or 10 nM pC5b6 with 10 nM C7 ( black circles ). After 30 minutes, bacteria were treated with 10 µg/ml trypsin for 20 minutes ( open blue squares and open red circles ). Trypsin was inhibited with 50 µg/ml soy-bean trypsin inhibitor and 10 nM C8, 100 nM C9-Cy5 and Sytox were added to complete MAC pores for 20 minutes after which bacteria were analyzed by flow cytometry. (B) Deposition of C9-Cy5 on bacteria treated as described in ( A ) was plotted as geoMFI of the bacterial population. ( C ) The percentage of bacteria with a damaged IM as determined by Sytox staining treated as described in ( A ). (D) Atomic force microscopy analysis (phase images) of E. coli MG1655 immobilized on Vectabond covered glass slides. Bacteria were treated as in ( A ) to label them with locally formed C5b-7 which was treated without or with trypsin before completing MAC pores by addition of C8 and C9. The concentrations of MAC components were ten-fold higher than described in ( A ). 2×2 µm 2 scans of whole bacteria were shown on top and representative 500×500 nm 2 zoomed images were shown on the bottom. Images are representative for a total of three bacteria per condition and at least four images per bacterium. Vertical scale bars: 12 deg for 2×2 µm 2 scans and 2 deg for 500×500 nm 2 scans. Data represent with mean +-SD ( B and C ) of at least 3 independent experiments. Statistical analysis was done using a paired one-way ANOVA with Tukey’s multiple comparisons’ test ( B ). Significance was shown as * p ≤ 0.05.

    Article Snippet: 100 µl of bacteria were immobilized on cleaned glass slides (Corning/Sigma Aldrich) covered with Vectabond® (Vector Laboratories, USA) as described previously for 30 minutes at RT ( ).

    Techniques: Derivative Assay, Purification, Incubation, Flow Cytometry, Staining, Microscopy

    Tapping mode atomic force microscopy phase images of MG1655 (A) and BL21 (B–C) E. coli bacteria immobilised onto glass coverslips. (A) When bacteria, on Vectabond® coated coverslips in HEPES buffer, are imaged at high resolution, a network of porins can be seen in the outer membrane. (B) AFM can be used to investigate the mechanism of action of antimicrobial peptides. As an example, 5 μM Cecropin B was applied to bacteria immobilised onto Vectabond® coated coverslips in HEPES buffer, resulting in nanometre-scale poration of the outer membrane. (C) Using cells immobilised on PLL in HEPES buffer, the formation of the membrane attack complex (MAC) can be investigated on live bacteria. The MAC pores can be observed as rings in the membrane. (A–C) Lateral scale bar is 100 nm. Vertical colour scale is (A) 2° (B) 4° and (C) 3°. (A–B) are 512 × 512 pixels, (C) is 256 × 256 pixels.

    Journal: The Analyst

    Article Title: Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

    doi: 10.1039/c9an01185d

    Figure Lengend Snippet: Tapping mode atomic force microscopy phase images of MG1655 (A) and BL21 (B–C) E. coli bacteria immobilised onto glass coverslips. (A) When bacteria, on Vectabond® coated coverslips in HEPES buffer, are imaged at high resolution, a network of porins can be seen in the outer membrane. (B) AFM can be used to investigate the mechanism of action of antimicrobial peptides. As an example, 5 μM Cecropin B was applied to bacteria immobilised onto Vectabond® coated coverslips in HEPES buffer, resulting in nanometre-scale poration of the outer membrane. (C) Using cells immobilised on PLL in HEPES buffer, the formation of the membrane attack complex (MAC) can be investigated on live bacteria. The MAC pores can be observed as rings in the membrane. (A–C) Lateral scale bar is 100 nm. Vertical colour scale is (A) 2° (B) 4° and (C) 3°. (A–B) are 512 × 512 pixels, (C) is 256 × 256 pixels.

    Article Snippet: Vectabond® Cleaned coverslips were put into a rack and submerged in 50 mL acetone for 5 minutes, then moved to a 50 : 1 solution of acetone : Vectabond® (Vector Laboratories, USA) for 5 minutes.

    Techniques: Microscopy

    Representative brightfield and fluorescence microscopy images of E. coli cells (BL21 and MG1655) immobilised on Vectabond® coated coverslips under different buffer conditions. Fluorescent bacteria are labelled with SYTOX® green dead cell stain.

    Journal: The Analyst

    Article Title: Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

    doi: 10.1039/c9an01185d

    Figure Lengend Snippet: Representative brightfield and fluorescence microscopy images of E. coli cells (BL21 and MG1655) immobilised on Vectabond® coated coverslips under different buffer conditions. Fluorescent bacteria are labelled with SYTOX® green dead cell stain.

    Article Snippet: Vectabond® Cleaned coverslips were put into a rack and submerged in 50 mL acetone for 5 minutes, then moved to a 50 : 1 solution of acetone : Vectabond® (Vector Laboratories, USA) for 5 minutes.

    Techniques: Fluorescence, Microscopy, Staining