goat anti mouse igg  (Thermo Fisher)


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    Goat anti Mouse IgG
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    PA1-32125
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    Thermo Fisher goat anti mouse igg
    Detection of MUL_3720 in M. ulcerans infected mouse foot pads. Antigen capture sandwich ELISAs using <t>mAb</t> JD3.4 in combination with polyclonal anti-MUL_3720 rabbit <t>IgG</t> as capturing and detecting antibodies, respectively, were carried out to detect MUL_3720 in lysates of mouse foot pads infected with M. ulcerans . Lysates of non-infected mouse foot pads served as negative control. Purified recombinant MUL_3720 was included as positive control. Asterisks indicate significant differences (P

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    1) Product Images from "Identification of the Mycobacterium ulcerans Protein MUL_3720 as a Promising Target for the Development of a Diagnostic Test for Buruli Ulcer"

    Article Title: Identification of the Mycobacterium ulcerans Protein MUL_3720 as a Promising Target for the Development of a Diagnostic Test for Buruli Ulcer

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0003477

    Detection of MUL_3720 in M. ulcerans infected mouse foot pads. Antigen capture sandwich ELISAs using mAb JD3.4 in combination with polyclonal anti-MUL_3720 rabbit IgG as capturing and detecting antibodies, respectively, were carried out to detect MUL_3720 in lysates of mouse foot pads infected with M. ulcerans . Lysates of non-infected mouse foot pads served as negative control. Purified recombinant MUL_3720 was included as positive control. Asterisks indicate significant differences (P
    Figure Legend Snippet: Detection of MUL_3720 in M. ulcerans infected mouse foot pads. Antigen capture sandwich ELISAs using mAb JD3.4 in combination with polyclonal anti-MUL_3720 rabbit IgG as capturing and detecting antibodies, respectively, were carried out to detect MUL_3720 in lysates of mouse foot pads infected with M. ulcerans . Lysates of non-infected mouse foot pads served as negative control. Purified recombinant MUL_3720 was included as positive control. Asterisks indicate significant differences (P

    Techniques Used: Infection, Negative Control, Purification, Recombinant, Positive Control

    Reactivity of MUL_3720-specific antibodies. Mouse mAbs JD3.2 (1), JD3.3 (2), JD3.4 (3), JD3.6 (4) and JD3.7 (5) and rabbit polyclonal IgG SZ3398 (6) and SZ3403 (7) were tested for their reactivity with MUL_3720 (aa 1–207) ( A ), truncated MUL_3720 (aa 115–207) ( B ) and the endogenous MUL_3720 in a total protein lysate of M. ulcerans strain NM20/02 ( C ) by Western Blot analysis.
    Figure Legend Snippet: Reactivity of MUL_3720-specific antibodies. Mouse mAbs JD3.2 (1), JD3.3 (2), JD3.4 (3), JD3.6 (4) and JD3.7 (5) and rabbit polyclonal IgG SZ3398 (6) and SZ3403 (7) were tested for their reactivity with MUL_3720 (aa 1–207) ( A ), truncated MUL_3720 (aa 115–207) ( B ) and the endogenous MUL_3720 in a total protein lysate of M. ulcerans strain NM20/02 ( C ) by Western Blot analysis.

    Techniques Used: Western Blot

    Sandwich ELISA capturing MUL_3720. Antigen capture sandwich ELISAs using mAb JD3.4 in combination with polyclonal anti-MUL_3720 rabbit IgG as capturing and detecting antibodies, respectively, were carried out to detect serial dilutions of recombinant MUL_3720 (A) and the endogenous MUL_3720 present in M. ulcerans lysates (B) .
    Figure Legend Snippet: Sandwich ELISA capturing MUL_3720. Antigen capture sandwich ELISAs using mAb JD3.4 in combination with polyclonal anti-MUL_3720 rabbit IgG as capturing and detecting antibodies, respectively, were carried out to detect serial dilutions of recombinant MUL_3720 (A) and the endogenous MUL_3720 present in M. ulcerans lysates (B) .

    Techniques Used: Sandwich ELISA, Recombinant

    2) Product Images from "Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice"

    Article Title: Systemic treatment with CAR-engineered T cells against PSCA delays subcutaneous tumor growth and prolongs survival of mice

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-30

    Lentivirus cassette and surface expression of PSCA-CAR on T cells. (A) The design of the PSCA-CAR-encoding lentiviral vector is shown. (B) Expression of the PSCA-CAR molecule on the surface CD3 + T cells after transduction with the lentiviral vector was analyzed by flow cytometry using Alexa-647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L). The solid filled histogram represents PSCA-CAR expression of transduced T cells, complex histogram represents CAR expression on Mock lentivirus-transduced T cells and the dashed histogram represents untransduced control T cells.
    Figure Legend Snippet: Lentivirus cassette and surface expression of PSCA-CAR on T cells. (A) The design of the PSCA-CAR-encoding lentiviral vector is shown. (B) Expression of the PSCA-CAR molecule on the surface CD3 + T cells after transduction with the lentiviral vector was analyzed by flow cytometry using Alexa-647 F(ab')2 Fragment of Goat Anti-Mouse IgG (H + L). The solid filled histogram represents PSCA-CAR expression of transduced T cells, complex histogram represents CAR expression on Mock lentivirus-transduced T cells and the dashed histogram represents untransduced control T cells.

    Techniques Used: Expressing, Plasmid Preparation, Transduction, Flow Cytometry, Cytometry

    3) Product Images from "Aminopeptidase N (CD13) functionally interacts with FcγRs in human monocytes"

    Article Title: Aminopeptidase N (CD13) functionally interacts with FcγRs in human monocytes

    Journal: Journal of Leukocyte Biology

    doi: 10.1189/jlb.1204714

    CD13 redistributes to the phagocytic cup during FcγR‐mediated phagocytosis in U‐937 cells. (A) CD13 distribution on resting cells. (B–D) U‐937 cells were incubated with IgG‐opsonized erythrocytes for 60 min at 37°C. Cells were fixed, and CD13 was detected by incubation with TR‐labeled F(ab)′ 2 fragments of mAb anti‐CD13. (D) Redistributed CD13 in the zone of contact with erythrocytes. (E–H) U‐937 cells were incubated with FITC‐IgG‐opsonized erythrocytes for 60 min at 37°C. Cells were chilled, and CD13 was detected by incubation with TR‐labeled F(ab)′ 2 fragments of mAb anti‐CD13. CD13 is visualized in the zones of the membrane in contact with erythrocytes (E, white arrows). Cell membranes are out of focus (see transmitted light, F), impeding detection of distribution of the rest of the population of CD13 molecules in this slice. Erythrocytes are fluorescent in the green channel (G). (H) Projection shows in yellow sites of colocalization of FcγR (IgG‐opsonized erythrocytes) and CD13 (arrows). (I–L) U‐937 cells were incubated with TR‐labeled F(ab)′ 2 fragments of anti‐CD13 mAb at 4°C before phagocytosis, washed, and incubated with FITC‐IgG‐opsonized erythrocytes for 90 min at 37°C. Noninternalized erythrocytes were lysed before observation in the confocal microscope. Erythrocytes internalized into phagosomes (J, K) are surrounded by CD13 (I). (J and L) Colocalization of the CD13‐red signal with FITC‐IgG‐labeled erythrocytes in the phagosomes. (M–O) Similar experiment as that shown in I–L but with erythrocytes opsonized with nonlabeled IgG. CD13 is seen inside the phagosomes (M). No fluorescence is seen in the green channel (O). (P) Transmitted light and red channel‐composed image of U‐937 cells incubated with TR‐labeled F(ab)′ 2 fragments of anti‐CD13 mAb before incubation with IgG‐opsonized erythrocytes for 60 min at 4°C. CD13 does not redistribute to zones of contact with erythrocytes.
    Figure Legend Snippet: CD13 redistributes to the phagocytic cup during FcγR‐mediated phagocytosis in U‐937 cells. (A) CD13 distribution on resting cells. (B–D) U‐937 cells were incubated with IgG‐opsonized erythrocytes for 60 min at 37°C. Cells were fixed, and CD13 was detected by incubation with TR‐labeled F(ab)′ 2 fragments of mAb anti‐CD13. (D) Redistributed CD13 in the zone of contact with erythrocytes. (E–H) U‐937 cells were incubated with FITC‐IgG‐opsonized erythrocytes for 60 min at 37°C. Cells were chilled, and CD13 was detected by incubation with TR‐labeled F(ab)′ 2 fragments of mAb anti‐CD13. CD13 is visualized in the zones of the membrane in contact with erythrocytes (E, white arrows). Cell membranes are out of focus (see transmitted light, F), impeding detection of distribution of the rest of the population of CD13 molecules in this slice. Erythrocytes are fluorescent in the green channel (G). (H) Projection shows in yellow sites of colocalization of FcγR (IgG‐opsonized erythrocytes) and CD13 (arrows). (I–L) U‐937 cells were incubated with TR‐labeled F(ab)′ 2 fragments of anti‐CD13 mAb at 4°C before phagocytosis, washed, and incubated with FITC‐IgG‐opsonized erythrocytes for 90 min at 37°C. Noninternalized erythrocytes were lysed before observation in the confocal microscope. Erythrocytes internalized into phagosomes (J, K) are surrounded by CD13 (I). (J and L) Colocalization of the CD13‐red signal with FITC‐IgG‐labeled erythrocytes in the phagosomes. (M–O) Similar experiment as that shown in I–L but with erythrocytes opsonized with nonlabeled IgG. CD13 is seen inside the phagosomes (M). No fluorescence is seen in the green channel (O). (P) Transmitted light and red channel‐composed image of U‐937 cells incubated with TR‐labeled F(ab)′ 2 fragments of anti‐CD13 mAb before incubation with IgG‐opsonized erythrocytes for 60 min at 4°C. CD13 does not redistribute to zones of contact with erythrocytes.

    Techniques Used: Incubation, Labeling, Microscopy, Fluorescence

    4) Product Images from "DAP12 Stabilizes the C-terminal Fragment of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) and Protects against LPS-induced Pro-inflammatory Response *"

    Article Title: DAP12 Stabilizes the C-terminal Fragment of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) and Protects against LPS-induced Pro-inflammatory Response *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.645986

    Effects of DAP12 on TREM2-CTF require their interaction. A , HEK293T cells were co-transfected with TREM2-Myc and wild-type (DAP12 WT-GFP) or mutant (DAP12 D50A-GFP) DAP12 plasmids. Lysates were immunoprecipitated using antibodies against mouse IgG (M
    Figure Legend Snippet: Effects of DAP12 on TREM2-CTF require their interaction. A , HEK293T cells were co-transfected with TREM2-Myc and wild-type (DAP12 WT-GFP) or mutant (DAP12 D50A-GFP) DAP12 plasmids. Lysates were immunoprecipitated using antibodies against mouse IgG (M

    Techniques Used: Transfection, Mutagenesis, Immunoprecipitation

    5) Product Images from "The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme"

    Article Title: The Cytoplasmic Capping Complex Assembles on Adapter Protein Nck1 Bound to the Proline-Rich C-Terminus of Mammalian Capping Enzyme

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001933

    Identification of Nck1 as a component of the cytoplasmic capping complex. (A) Cytoplasmic extract from cells expressing bio-cCE was separated on a 10%–50% glycerol gradient. Fractions containing each of these proteins were identified by Western blotting of input fractions with antibodies to the Myc tag on bio-cCE and to Nck1 (upper 2 panels). Streptavidin beads were used to recover bio-cCE from individual fractions and bound proteins were again analyzed by Western blotting with anti-Myc and anti-Nck1 antibodies (lower two panels). (B) Cytoplasmic extract from non-transfected cells was separated on a calibrated Sephacryl S-200 column. Starting with the void volume individual fractions were collected and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The missing CE band in fraction 3 was due to sample loss during loading. (C) The fractions indicated with a box at the bottom of (B) were pooled and immunoprecipitated with anti-Nck1 or control IgG. 20% of the immunoprecipitated sample was used for Western blotting with anti-Nck1 antibody and 70% of the immunoprecipitated sample was used for Western blotting with anti-CE antibody. (D) Cytoplasmic extract from non-transfected cells was immunoprecipitated with anti-CE antibody or control IgG, and the recovered proteins were analyzed by Western blotting with anti-Nck1 antibody.
    Figure Legend Snippet: Identification of Nck1 as a component of the cytoplasmic capping complex. (A) Cytoplasmic extract from cells expressing bio-cCE was separated on a 10%–50% glycerol gradient. Fractions containing each of these proteins were identified by Western blotting of input fractions with antibodies to the Myc tag on bio-cCE and to Nck1 (upper 2 panels). Streptavidin beads were used to recover bio-cCE from individual fractions and bound proteins were again analyzed by Western blotting with anti-Myc and anti-Nck1 antibodies (lower two panels). (B) Cytoplasmic extract from non-transfected cells was separated on a calibrated Sephacryl S-200 column. Starting with the void volume individual fractions were collected and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The missing CE band in fraction 3 was due to sample loss during loading. (C) The fractions indicated with a box at the bottom of (B) were pooled and immunoprecipitated with anti-Nck1 or control IgG. 20% of the immunoprecipitated sample was used for Western blotting with anti-Nck1 antibody and 70% of the immunoprecipitated sample was used for Western blotting with anti-CE antibody. (D) Cytoplasmic extract from non-transfected cells was immunoprecipitated with anti-CE antibody or control IgG, and the recovered proteins were analyzed by Western blotting with anti-Nck1 antibody.

    Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation

    6) Product Images from "Retinoic Acid-Loaded PLGA Nanoparticle Formulation of ApoB-100-Derived Peptide 210 Attenuates Atherosclerosis"

    Article Title: Retinoic Acid-Loaded PLGA Nanoparticle Formulation of ApoB-100-Derived Peptide 210 Attenuates Atherosclerosis

    Journal: Journal of biomedical nanotechnology

    doi: 10.1166/jbn.2020.2905

    Titers of P210-specific total IgM or IgG from immunized Apoe −/− mice. Plasma from Apoe −/− mice was obtained before immunization (pre-immunization) and at termination (post-immunization). A. Titers of P210-specific total IgM. B. Titers of IgM against MDA-modified P210. C. The values of P210-specific IgM titers differed between post-immunization and pre-immunization. D. The values of MDA-modified P210-specific IgM titers differed between post-immunization and pre-immunization. E. Titers of P210-specific IgG. F. Titers of IgG against MDA-modified P210. Data represent arithmetic means ± SEM of specific antibodies. All the statistical analysis of experiments comparing the five groups was evaluated by one-way ANOVA.
    Figure Legend Snippet: Titers of P210-specific total IgM or IgG from immunized Apoe −/− mice. Plasma from Apoe −/− mice was obtained before immunization (pre-immunization) and at termination (post-immunization). A. Titers of P210-specific total IgM. B. Titers of IgM against MDA-modified P210. C. The values of P210-specific IgM titers differed between post-immunization and pre-immunization. D. The values of MDA-modified P210-specific IgM titers differed between post-immunization and pre-immunization. E. Titers of P210-specific IgG. F. Titers of IgG against MDA-modified P210. Data represent arithmetic means ± SEM of specific antibodies. All the statistical analysis of experiments comparing the five groups was evaluated by one-way ANOVA.

    Techniques Used: Mouse Assay, Multiple Displacement Amplification, Modification

    7) Product Images from "Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)"

    Article Title: Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17086-8

    Pre-labeling Ex- d STORM. a Simulated intensity profiles using a cylindrical distribution function to describe unexpanded or 3.2x expanded immunostained microtubules (labeled with IgG antibodies or DNA modified IgG antibodies pre-expansion) and resulting peak-to-peak distances of the cross-sectional profiles. b d STORM image of expanded and re-embedded α- and β-tubulin pre-labeled with secondary Alexa Fluor 532 IgG antibodies (Al532) using the MA-NHS/GA method 6 , i.e. antibodies are cross-linked with glutaraldehyde (GA) into the hydrogel (Antibody-Al532 (GA)). c Zoom in of white boxed region in ( b ). d Averaged cross-sectional profile of 8 microtubule segments with a length between 1.5–6.4 µm and 28.6 µm in total measured in 4 expanded cells. e Histogram of peak-to-peak distance distribution with normalized normal curve (red) of microtubule segments analyzed in ( d ) at n = 8 microtubule segments in 4 cells from 1 expansion experiment with a mean distance of 133.8 ± 13.2 nm (mean ± sd). f Unexpanded d STORM image of ssDNA-Cy5 secondary antibody hybridized with Cy5 bearing oligonucleotides pre-expansion (DNA-Cy5 protocol). g Magnified view of white boxed region in ( f ). h Average cross-sectional profile of 7 microtubule segments with a length between 1.0–1.8 µm and 8.7 µm in total. i Histogram of peak-to-peak distances with normalized normal distribution curve (red) of the data analyzed in ( h ) along n = 7 microtubule segments in 2 cells from 1 experiment with a mean distance of 43.9 ± 3.7 nm (mean ± sd). j Expanded d STORM image of microtubules labeled with α-tubulin and dsDNA (DNA-Al532) conjugated secondary antibodies exhibiting a methacryloyl group to crosslink the DNA with fluorophores pre-expansion into the hydrogel (original ExM trifunctional label concept) 1 . k Zoom-in of white boxed region in ( j ). l Average intensity profile of 26 microtubule segments with a length of 2.4–10.7 µm and 118.6 µm in total. m Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined from n = 26 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 226.7 ± 15.3 nm (mean ± sd). n d STORM image of α- and β-tubulin expanded according to the DNA-Cy5 protocol strategy with labels at Cy5-bearing oligonucleotides introduced post-re-embedding. o Zoom in of white boxed region in ( n ). p Average intensity profile of 15 microtubule segments with a length between 1.6–25.1 µm and a total length of 126.0 µm in 1 expanded sample. q Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by fitting the cross-sectional profiles analyzed in ( p ) along n = 22 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 201.0 ± 12.9 nm (mean ± sd). The small logos in the upper left corner symbolize the labeling method, e.g. pre- and post-immunolabeled with or without DNA-linker, respectively. Scale bars, 2 µm ( b , f , j , n ), 500 nm ( c , g , k , o ).
    Figure Legend Snippet: Pre-labeling Ex- d STORM. a Simulated intensity profiles using a cylindrical distribution function to describe unexpanded or 3.2x expanded immunostained microtubules (labeled with IgG antibodies or DNA modified IgG antibodies pre-expansion) and resulting peak-to-peak distances of the cross-sectional profiles. b d STORM image of expanded and re-embedded α- and β-tubulin pre-labeled with secondary Alexa Fluor 532 IgG antibodies (Al532) using the MA-NHS/GA method 6 , i.e. antibodies are cross-linked with glutaraldehyde (GA) into the hydrogel (Antibody-Al532 (GA)). c Zoom in of white boxed region in ( b ). d Averaged cross-sectional profile of 8 microtubule segments with a length between 1.5–6.4 µm and 28.6 µm in total measured in 4 expanded cells. e Histogram of peak-to-peak distance distribution with normalized normal curve (red) of microtubule segments analyzed in ( d ) at n = 8 microtubule segments in 4 cells from 1 expansion experiment with a mean distance of 133.8 ± 13.2 nm (mean ± sd). f Unexpanded d STORM image of ssDNA-Cy5 secondary antibody hybridized with Cy5 bearing oligonucleotides pre-expansion (DNA-Cy5 protocol). g Magnified view of white boxed region in ( f ). h Average cross-sectional profile of 7 microtubule segments with a length between 1.0–1.8 µm and 8.7 µm in total. i Histogram of peak-to-peak distances with normalized normal distribution curve (red) of the data analyzed in ( h ) along n = 7 microtubule segments in 2 cells from 1 experiment with a mean distance of 43.9 ± 3.7 nm (mean ± sd). j Expanded d STORM image of microtubules labeled with α-tubulin and dsDNA (DNA-Al532) conjugated secondary antibodies exhibiting a methacryloyl group to crosslink the DNA with fluorophores pre-expansion into the hydrogel (original ExM trifunctional label concept) 1 . k Zoom-in of white boxed region in ( j ). l Average intensity profile of 26 microtubule segments with a length of 2.4–10.7 µm and 118.6 µm in total. m Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined from n = 26 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 226.7 ± 15.3 nm (mean ± sd). n d STORM image of α- and β-tubulin expanded according to the DNA-Cy5 protocol strategy with labels at Cy5-bearing oligonucleotides introduced post-re-embedding. o Zoom in of white boxed region in ( n ). p Average intensity profile of 15 microtubule segments with a length between 1.6–25.1 µm and a total length of 126.0 µm in 1 expanded sample. q Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by fitting the cross-sectional profiles analyzed in ( p ) along n = 22 microtubule segments in 4 cells from 1 expanded sample showing a mean distance of 201.0 ± 12.9 nm (mean ± sd). The small logos in the upper left corner symbolize the labeling method, e.g. pre- and post-immunolabeled with or without DNA-linker, respectively. Scale bars, 2 µm ( b , f , j , n ), 500 nm ( c , g , k , o ).

    Techniques Used: Labeling, Modification, Immunolabeling

    Re-embedding enables Ex- d STORM. a Model of microtubules with an outer diameter of 25 nm stained with conventional primary (pab) and fluorescently labeled secondary IgG antibodies (sab) results in a total diameter of 60 nm with a linkage error (defined by the size of the primary and secondary antibody) of 17.5 nm 22 . b d STORM image of pre-labeled proExM expanded and re-embedded Cos-7 cells stained with primary antibodies against α-tubulin and secondary Alexa Fluor 532 conjugated antibodies (Al532). The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion (pre-labeled). c Zoom in on highlighted region in ( b ). d Averaged cross-sectional profile of nine microtubule segments with a total length of 29.1 µm (segment lengths range from 2.1-4.5 µm) measured in two cells from 1 expanded sample. e Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by bi-Gaussian fitting of the data analyzed in ( c ) with an average distance of 137.1 ± 10.1 nm (mean ± sd). The data were obtained from n = 9 microtubule segments in 2 cells from 1 expanded sample. f Unexpanded Cos-7 cells labeled with an anti α-tubulin primary antibody and Alexa Fluor 532 (Al532) conjugated IgG secondary antibodies. The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded. g Zoom in of the white boxed region in ( f ). h Average intensity profile of 35 microtubule segments with a length between 1.1 and 5.8 µm (mean = 2.0 µm) and a total length of 69.6 µm analyzed in 12 d STORM images. i Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by bi-Gaussian fitting of cross-sectional profiles of the analyzed microtubule segments in ( h ) with a mean peak-to-peak distance of 36.2 ± 5.4 nm (mean ± sd). The data were obtained from n =35 microtubule segments in 12 cells and 3 independent experiments. Scale bars, 2 µm ( b , f ), 500 nm ( c , g ).
    Figure Legend Snippet: Re-embedding enables Ex- d STORM. a Model of microtubules with an outer diameter of 25 nm stained with conventional primary (pab) and fluorescently labeled secondary IgG antibodies (sab) results in a total diameter of 60 nm with a linkage error (defined by the size of the primary and secondary antibody) of 17.5 nm 22 . b d STORM image of pre-labeled proExM expanded and re-embedded Cos-7 cells stained with primary antibodies against α-tubulin and secondary Alexa Fluor 532 conjugated antibodies (Al532). The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion (pre-labeled). c Zoom in on highlighted region in ( b ). d Averaged cross-sectional profile of nine microtubule segments with a total length of 29.1 µm (segment lengths range from 2.1-4.5 µm) measured in two cells from 1 expanded sample. e Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by bi-Gaussian fitting of the data analyzed in ( c ) with an average distance of 137.1 ± 10.1 nm (mean ± sd). The data were obtained from n = 9 microtubule segments in 2 cells from 1 expanded sample. f Unexpanded Cos-7 cells labeled with an anti α-tubulin primary antibody and Alexa Fluor 532 (Al532) conjugated IgG secondary antibodies. The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded. g Zoom in of the white boxed region in ( f ). h Average intensity profile of 35 microtubule segments with a length between 1.1 and 5.8 µm (mean = 2.0 µm) and a total length of 69.6 µm analyzed in 12 d STORM images. i Histogram of peak-to-peak distances with normalized normal distribution curve (red) determined by bi-Gaussian fitting of cross-sectional profiles of the analyzed microtubule segments in ( h ) with a mean peak-to-peak distance of 36.2 ± 5.4 nm (mean ± sd). The data were obtained from n =35 microtubule segments in 12 cells and 3 independent experiments. Scale bars, 2 µm ( b , f ), 500 nm ( c , g ).

    Techniques Used: Staining, Labeling, Immunolabeling

    8) Product Images from "CD1c molecules broadly survey the endocytic system"

    Article Title: CD1c molecules broadly survey the endocytic system

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Colocalization of CD1c with ARF6-T27N. HeLa cell transfectants expressing CD1c were grown on coverslips and transiently supertransfected with ARF6-T27N cDNA. The cells were then fixed and permeabilized, and double-labeling with mouse anti-CD1c antibody (detected with Texas Red-conjugated donkey anti-mouse IgG) and rabbit anti-ARF6 antibody (detected with FITC-conjugated donkey anti-rabbit IgG) was performed. Fluorescent confocal images were obtained for CD1c ( A ) and ARF6 ( B ). The two images then were superimposed to detect vesicles expressing both CD1c and ARF6 ( C , yellow vesicles shown with arrowheads). (Scale bars = 5 μm.)
    Figure Legend Snippet: Colocalization of CD1c with ARF6-T27N. HeLa cell transfectants expressing CD1c were grown on coverslips and transiently supertransfected with ARF6-T27N cDNA. The cells were then fixed and permeabilized, and double-labeling with mouse anti-CD1c antibody (detected with Texas Red-conjugated donkey anti-mouse IgG) and rabbit anti-ARF6 antibody (detected with FITC-conjugated donkey anti-rabbit IgG) was performed. Fluorescent confocal images were obtained for CD1c ( A ) and ARF6 ( B ). The two images then were superimposed to detect vesicles expressing both CD1c and ARF6 ( C , yellow vesicles shown with arrowheads). (Scale bars = 5 μm.)

    Techniques Used: Expressing, Labeling

    Colocalization of CD1a, -b, and -c with LAMP-1. Monocyte-derived dendritic cells were fixed and permeablized after a cytospin procedure. The cells were then double-labeled with mouse mAbs to CD1a ( A ), CD1b ( D ), or CD1c ( G ) (detected with Texas Red-conjugated donkey anti-mouse IgG) and a rabbit antiserum against human LAMP-1 ( B , E , and H ) (detected with FITC-conjugated donkey anti-rabbit IgG) and analyzed by confocal microscopy. The corresponding red and green fluorescent confocal images then were superimposed to detect any cellular compartments expressing both CD1 and LAMP-1 ( C , F , and I , yellow vesicles). (Scale bars = 5 μm.)
    Figure Legend Snippet: Colocalization of CD1a, -b, and -c with LAMP-1. Monocyte-derived dendritic cells were fixed and permeablized after a cytospin procedure. The cells were then double-labeled with mouse mAbs to CD1a ( A ), CD1b ( D ), or CD1c ( G ) (detected with Texas Red-conjugated donkey anti-mouse IgG) and a rabbit antiserum against human LAMP-1 ( B , E , and H ) (detected with FITC-conjugated donkey anti-rabbit IgG) and analyzed by confocal microscopy. The corresponding red and green fluorescent confocal images then were superimposed to detect any cellular compartments expressing both CD1 and LAMP-1 ( C , F , and I , yellow vesicles). (Scale bars = 5 μm.)

    Techniques Used: Derivative Assay, Labeling, Confocal Microscopy, Expressing

    9) Product Images from "The oncomir face of microRNA-206: A permanent miR-206 transfection study"

    Article Title: The oncomir face of microRNA-206: A permanent miR-206 transfection study

    Journal: Experimental Biology and Medicine

    doi: 10.1177/1535370218795406

    SMARCB1 immunocytochemistry and flow cytometry analysis of SS-iASC and SS-iASC-206. (a) Positive SMARCB1 staining of nuclei of not transfected SS-iASC cell line. (b and c) Between the strongly SMARCB1-immunopositive nuclei, decreased SMARCB1 protein expression was observed in individual nuclei of SS-iASC-206; (*) marks the decreased nuclear SMARCB1 expression. (d) Measurement of SMARCB1 expression by flow cytometry: gating strategy on the living cells using forward and side scattered light scattergram. (e) Fluorescence intensity of SMARCB1 in SS-iASC-206 and SS-iASC cell populations labeled with Alexa Flour® 647 conjugated goat anti-mouse IgG. The red histogram demonstrates miR-206 transfected SS-iASC-206, while the blue histogram shows the not transfected SS-iASC population. (A color version of this figure is available in the online journal.)
    Figure Legend Snippet: SMARCB1 immunocytochemistry and flow cytometry analysis of SS-iASC and SS-iASC-206. (a) Positive SMARCB1 staining of nuclei of not transfected SS-iASC cell line. (b and c) Between the strongly SMARCB1-immunopositive nuclei, decreased SMARCB1 protein expression was observed in individual nuclei of SS-iASC-206; (*) marks the decreased nuclear SMARCB1 expression. (d) Measurement of SMARCB1 expression by flow cytometry: gating strategy on the living cells using forward and side scattered light scattergram. (e) Fluorescence intensity of SMARCB1 in SS-iASC-206 and SS-iASC cell populations labeled with Alexa Flour® 647 conjugated goat anti-mouse IgG. The red histogram demonstrates miR-206 transfected SS-iASC-206, while the blue histogram shows the not transfected SS-iASC population. (A color version of this figure is available in the online journal.)

    Techniques Used: Immunocytochemistry, Flow Cytometry, Cytometry, Staining, Transfection, Expressing, Fluorescence, Labeling

    10) Product Images from "Conformational fingerprint of blood and tissue ACEs: Personalized approach"

    Article Title: Conformational fingerprint of blood and tissue ACEs: Personalized approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209861

    Enzyme (ACE) immune-capture assay. A . Scheme of the method. Goat anti-mouse IgG are loaded on the 96-wells plate to minimize non-specific adsorption of both mAbs and ACE, as well as prevent putative denaturation of some mAbs as a result of a contact with plastic. After washing of unbound anti-mouse IgG, mAbs to ACE are applied to the plate and, after another washing, analyzed ACE source is added. The amount of ACE in complex with any mAb is estimated by measuring precipitated ACE activity towards specific substrate, usually ZPHL, added directly into the wells. B . The dependence of the fluorescence (reflecting relative ACE activity in wells) on the loaded ACE activity on wells covered by strong mAb 9B9 (10 μg/ml) and weak mAb 1E10 (10 μg/ml). C . The dependence of the fluorescence signal on the loaded ACE activity at different concentrations of the loaded strong mAb 9B9. D . The dependence of the fluorescence signal on the loaded ACE activity at different concentrations of the loaded weak mAb 1E10.
    Figure Legend Snippet: Enzyme (ACE) immune-capture assay. A . Scheme of the method. Goat anti-mouse IgG are loaded on the 96-wells plate to minimize non-specific adsorption of both mAbs and ACE, as well as prevent putative denaturation of some mAbs as a result of a contact with plastic. After washing of unbound anti-mouse IgG, mAbs to ACE are applied to the plate and, after another washing, analyzed ACE source is added. The amount of ACE in complex with any mAb is estimated by measuring precipitated ACE activity towards specific substrate, usually ZPHL, added directly into the wells. B . The dependence of the fluorescence (reflecting relative ACE activity in wells) on the loaded ACE activity on wells covered by strong mAb 9B9 (10 μg/ml) and weak mAb 1E10 (10 μg/ml). C . The dependence of the fluorescence signal on the loaded ACE activity at different concentrations of the loaded strong mAb 9B9. D . The dependence of the fluorescence signal on the loaded ACE activity at different concentrations of the loaded weak mAb 1E10.

    Techniques Used: Adsorption, Activity Assay, Fluorescence

    Effect of plate washing on the dissociation of ACE inhibitor from ACE. The residual ACE activity towards substrates, HHL and ZPHL, as well the ratio of the rates of the hydrolysis of these substrates, ZPHL/HHL ratio, were determined in the wells coated by goat anti-mouse IgG, four mAbs to ACE (strong and weak) and, then, the mixture of purified seminal fluid ACE with inhibitor enalaprilat (100 nM) after different number of washings with water with 0.05% (v/v) Tween 20. The ZPHL/HHL ratio for ACE in solution in the presence of the inhibitor (red bar in the right column) is shown for comparison. Data are expressed as a % of control (i.e. ACE without inhibitor) and presented as a mean of at least 3 independent experiments. The coloring here and in other figures is as follows: Values increased more than by 20% are highlighted in orange, more than 50% are highlighted in dark orange, more than by 100% are highlighted in red. Values decreased more than by 20% are highlighted in yellow.
    Figure Legend Snippet: Effect of plate washing on the dissociation of ACE inhibitor from ACE. The residual ACE activity towards substrates, HHL and ZPHL, as well the ratio of the rates of the hydrolysis of these substrates, ZPHL/HHL ratio, were determined in the wells coated by goat anti-mouse IgG, four mAbs to ACE (strong and weak) and, then, the mixture of purified seminal fluid ACE with inhibitor enalaprilat (100 nM) after different number of washings with water with 0.05% (v/v) Tween 20. The ZPHL/HHL ratio for ACE in solution in the presence of the inhibitor (red bar in the right column) is shown for comparison. Data are expressed as a % of control (i.e. ACE without inhibitor) and presented as a mean of at least 3 independent experiments. The coloring here and in other figures is as follows: Values increased more than by 20% are highlighted in orange, more than 50% are highlighted in dark orange, more than by 100% are highlighted in red. Values decreased more than by 20% are highlighted in yellow.

    Techniques Used: Activity Assay, Purification

    11) Product Images from "Polymorphic factor H-binding activity of CspA protects Lyme borreliae from the host complement in feeding ticks to facilitate tick-to-host transmission"

    Article Title: Polymorphic factor H-binding activity of CspA protects Lyme borreliae from the host complement in feeding ticks to facilitate tick-to-host transmission

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007106

    CspA variants differ in their ability in promoting spirochetes binding to FH from different vertebrate animals. B . burgdorferi strain B31-5A15 (“B31-5A15”), B31-5A4NP1Δ cspA harboring the vector pBSV2G (“Δ cspA /Vector”), or this cspA mutant strain producing CspA B31 (“Δ cspA /pCspA B31 ”), CspA PKo (“Δ cspA /pCspA PKo ”), CspA ZQ1 (“Δ cspA /pCspA ZQ1 ”), or CspA B31 L246D (“Δ cspA /pCspA B31 L246D”), or B313 carrying the vector pBSV2G (“B313/Vector”, negative control) was incubated with FH from human, mouse, horse, or quail. The bacteria were stained with a sheep anti-FH polyclonal IgG (for the spirochetes incubated with human, mouse, or horse FH) or a mouse anti-FH monoclonal antibody VIG8 (for the spirochetes incubated with quail FH) followed by an Alexa 647-conjugated donkey anti-sheep IgG or goat anti-mouse IgG prior to being applied to flow cytometry analysis. (Left panel) Representative histograms of flow cytometry analysis showing the levels of FH from (A) human, (B) mouse, (C) horse, or (D) quail binding to indicated B . burgdorferi strains. (Right panel) The levels of B . burgdorferi binding to FH from (A) human, (B) mouse, (C) horse, or (D) quail were measured by flow cytometry and presented as mean fluorescence index (MFI). Each bar represents the mean of three independent determinations ± SEM. Significant differences (P
    Figure Legend Snippet: CspA variants differ in their ability in promoting spirochetes binding to FH from different vertebrate animals. B . burgdorferi strain B31-5A15 (“B31-5A15”), B31-5A4NP1Δ cspA harboring the vector pBSV2G (“Δ cspA /Vector”), or this cspA mutant strain producing CspA B31 (“Δ cspA /pCspA B31 ”), CspA PKo (“Δ cspA /pCspA PKo ”), CspA ZQ1 (“Δ cspA /pCspA ZQ1 ”), or CspA B31 L246D (“Δ cspA /pCspA B31 L246D”), or B313 carrying the vector pBSV2G (“B313/Vector”, negative control) was incubated with FH from human, mouse, horse, or quail. The bacteria were stained with a sheep anti-FH polyclonal IgG (for the spirochetes incubated with human, mouse, or horse FH) or a mouse anti-FH monoclonal antibody VIG8 (for the spirochetes incubated with quail FH) followed by an Alexa 647-conjugated donkey anti-sheep IgG or goat anti-mouse IgG prior to being applied to flow cytometry analysis. (Left panel) Representative histograms of flow cytometry analysis showing the levels of FH from (A) human, (B) mouse, (C) horse, or (D) quail binding to indicated B . burgdorferi strains. (Right panel) The levels of B . burgdorferi binding to FH from (A) human, (B) mouse, (C) horse, or (D) quail were measured by flow cytometry and presented as mean fluorescence index (MFI). Each bar represents the mean of three independent determinations ± SEM. Significant differences (P

    Techniques Used: Binding Assay, Plasmid Preparation, Mutagenesis, Negative Control, Incubation, Staining, Flow Cytometry, Cytometry, Fluorescence

    CspA variants differ in their ability to reduce the deposition of C3b or MAC from different vertebrate animals on the spirochete surface. B . burgdorferi strain B31-5A15 (“B31-5A15”), B31-5A4NP1Δ cspA harboring the vector pBSV2G (“Δ cspA /Vector”), or this cspA mutant strain producing CspA B31 (“Δ cspA /pCspA B31 ”), CspA PKo (“Δ cspA /pCspA PKo ”), CspA ZQ1 (“Δ cspA /pCspA ZQ1 ”), or CspA B31 L246D (“Δ cspA /pCspA B31 L246D”), or B313 carrying the vector pBSV2G (“B313/Vector”, negative control) was incubated with serum from human, mouse, or horse with a final concentration of 20%. The bacteria were stained with a guinea pig anti-C3 polyclonal IgG, a mouse anti-C5b-9 monoclonal antibody aE11 (for spirochetes incubated with human or horse serum), or a rabbit anti-C5b-9 polyclonal IgG (for spirochetes incubated with mouse serum) followed by an Alexa 647-conjugated goat anti-guinea pig IgG or goat anti-mouse IgG, or goat anti-rabbit IgG prior to being applied to flow cytometry analysis. Representative histograms of flow cytometry analysis showing the deposition levels of mouse (A) C3b or (C) MAC on the surface of indicated B . burgdorferi strains. The deposition levels of (B) C3b or (D) MAC of indicated animals on the surface of B . burgdorferi were measured by flow cytometry and presented as mean fluorescence index (MFI). Each bar represents the mean of three independent determinations ± SEM. Significant differences (P
    Figure Legend Snippet: CspA variants differ in their ability to reduce the deposition of C3b or MAC from different vertebrate animals on the spirochete surface. B . burgdorferi strain B31-5A15 (“B31-5A15”), B31-5A4NP1Δ cspA harboring the vector pBSV2G (“Δ cspA /Vector”), or this cspA mutant strain producing CspA B31 (“Δ cspA /pCspA B31 ”), CspA PKo (“Δ cspA /pCspA PKo ”), CspA ZQ1 (“Δ cspA /pCspA ZQ1 ”), or CspA B31 L246D (“Δ cspA /pCspA B31 L246D”), or B313 carrying the vector pBSV2G (“B313/Vector”, negative control) was incubated with serum from human, mouse, or horse with a final concentration of 20%. The bacteria were stained with a guinea pig anti-C3 polyclonal IgG, a mouse anti-C5b-9 monoclonal antibody aE11 (for spirochetes incubated with human or horse serum), or a rabbit anti-C5b-9 polyclonal IgG (for spirochetes incubated with mouse serum) followed by an Alexa 647-conjugated goat anti-guinea pig IgG or goat anti-mouse IgG, or goat anti-rabbit IgG prior to being applied to flow cytometry analysis. Representative histograms of flow cytometry analysis showing the deposition levels of mouse (A) C3b or (C) MAC on the surface of indicated B . burgdorferi strains. The deposition levels of (B) C3b or (D) MAC of indicated animals on the surface of B . burgdorferi were measured by flow cytometry and presented as mean fluorescence index (MFI). Each bar represents the mean of three independent determinations ± SEM. Significant differences (P

    Techniques Used: Plasmid Preparation, Mutagenesis, Negative Control, Incubation, Concentration Assay, Staining, Flow Cytometry, Cytometry, Fluorescence

    12) Product Images from "Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses"

    Article Title: Modification to the Capsid of the Adenovirus Vector That Enhances Dendritic Cell Infection and Transgene-Specific Cellular Immune Responses

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.5.2572-2580.2004

    Humoral response to the β-gal transgene following immunization with RGD-modified Ad vector compared to a wild-type capsid Ad vector. C57BL/6 mice were infected with AdZ.F(RGD), AdZ, or AdNull at 10 8 or 10 9 PU/mouse by subcutaneous (footpad) administration. PBS-injected naive mice served as controls. Anti-β-gal total IgM (A and B) and IgG (C and D) antibodies were determined at 2 and 4 weeks by enzyme-linked immunosorbent assay. (A) IgM, AdZ; (B) IgM, AdZ.F(RGD); (C) IgG, AdZ; (D) IgG, AdZ.F(RGD). Data are shown as mean and SEM of four mice per group. The limit of detection is shown as a dashed line.
    Figure Legend Snippet: Humoral response to the β-gal transgene following immunization with RGD-modified Ad vector compared to a wild-type capsid Ad vector. C57BL/6 mice were infected with AdZ.F(RGD), AdZ, or AdNull at 10 8 or 10 9 PU/mouse by subcutaneous (footpad) administration. PBS-injected naive mice served as controls. Anti-β-gal total IgM (A and B) and IgG (C and D) antibodies were determined at 2 and 4 weeks by enzyme-linked immunosorbent assay. (A) IgM, AdZ; (B) IgM, AdZ.F(RGD); (C) IgG, AdZ; (D) IgG, AdZ.F(RGD). Data are shown as mean and SEM of four mice per group. The limit of detection is shown as a dashed line.

    Techniques Used: Modification, Plasmid Preparation, Mouse Assay, Infection, Injection, Enzyme-linked Immunosorbent Assay

    13) Product Images from "PBF509, an Adenosine A2A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders"

    Article Title: PBF509, an Adenosine A2A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.01200

    Immunoreactivity of A 2A R in the striatum of 6-OHDA-lesioned rats. (A) Immunoblot analysis of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. Striatal membranes were analyzed by SDS–PAGE and immunoblotted using a rabbit anti-TH polyclonal antibody (1 μg/ml), a mouse anti-A 2A R monoclonal antibody (1 μg/ml) and a rabbit anti-α-actinin polyclonal antibody (1 μg/ml). A HRP-conjugated anti-rabbit or anti-mouse IgG (1/30,000) was used as a secondary antibody. The immunoreactive bands were visualized by chemiluminescence. Load control used for quantitating was α-actinin. A representative blot for three different lesioned animals is shown. (B) Quantification of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. The immunoreactive bands corresponding to TH and A 2A R in each striatal hemisphere were normalized by α-actinin immunoreactivity. Data are expressed as percentage of the control (R) TH or A 2A R density ± SEM of three independent experiments. Asterisks indicate data significantly different from the control condition: ∗∗∗ P
    Figure Legend Snippet: Immunoreactivity of A 2A R in the striatum of 6-OHDA-lesioned rats. (A) Immunoblot analysis of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. Striatal membranes were analyzed by SDS–PAGE and immunoblotted using a rabbit anti-TH polyclonal antibody (1 μg/ml), a mouse anti-A 2A R monoclonal antibody (1 μg/ml) and a rabbit anti-α-actinin polyclonal antibody (1 μg/ml). A HRP-conjugated anti-rabbit or anti-mouse IgG (1/30,000) was used as a secondary antibody. The immunoreactive bands were visualized by chemiluminescence. Load control used for quantitating was α-actinin. A representative blot for three different lesioned animals is shown. (B) Quantification of TH and A 2A R density in striatal membranes from control (R) and 6-OHDA lesioned (L) striatal hemisphere. The immunoreactive bands corresponding to TH and A 2A R in each striatal hemisphere were normalized by α-actinin immunoreactivity. Data are expressed as percentage of the control (R) TH or A 2A R density ± SEM of three independent experiments. Asterisks indicate data significantly different from the control condition: ∗∗∗ P

    Techniques Used: SDS Page

    14) Product Images from "Genetic Adjuvants in Replicating Single-Cycle Adenovirus Vectors Amplify Systemic and Mucosal Immune Responses against HIV-1 Envelope"

    Article Title: Genetic Adjuvants in Replicating Single-Cycle Adenovirus Vectors Amplify Systemic and Mucosal Immune Responses against HIV-1 Envelope

    Journal: Vaccines

    doi: 10.3390/vaccines8010064

    Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intramuscular (IM) immunization. Groups of 10 female BALB/c mice were immunized with PBS or 10 10 vp of the indicated SC-Ads. Six weeks later, samples were collected for ELISA vs. clade C CN54 gp140 ( A ) , ( B ), and ( C ) six week ELISAs after a single high dose IM immunization. Mean +/− SEM is shown. ( A ) 1/2000 sera dilutions detecting IgG. ( B ) IgG ELISA for 1/35 dilution of vaginal wash samples. ( C ) IgA ELISA for 1/35 dilution of vaginal wash samples. * p
    Figure Legend Snippet: Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intramuscular (IM) immunization. Groups of 10 female BALB/c mice were immunized with PBS or 10 10 vp of the indicated SC-Ads. Six weeks later, samples were collected for ELISA vs. clade C CN54 gp140 ( A ) , ( B ), and ( C ) six week ELISAs after a single high dose IM immunization. Mean +/− SEM is shown. ( A ) 1/2000 sera dilutions detecting IgG. ( B ) IgG ELISA for 1/35 dilution of vaginal wash samples. ( C ) IgA ELISA for 1/35 dilution of vaginal wash samples. * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intranasal (IN) immunization. Groups of 10 female BALB/c mice were immunized with phosphate-buffered saline (PBS) or 10 9 vp of the indicated SC-Ads. Six weeks later, samples were collected for enzyme-linked immunosorbent assay (ELISA) vs. clade C CN54 gp140. ( A ) Sub-isotyping ELISA for the indicated samples at 1/200 dilution (low dilution used for low-sensitivity sub-isotyping kit). All IgG isotypes in the granulocyte macrophage colony-stimulating factor (GMCSF) and TcdA/B groups were significantly different than PBS by 2-way ANOVA. ( B ) ELISA OD450 levels are shown for 1/35 dilution of vaginal wash samples with detection by anti-IgA. * p
    Figure Legend Snippet: Effects of SC-Ad genetic adjuvants on clade C env antibody responses in mice after intranasal (IN) immunization. Groups of 10 female BALB/c mice were immunized with phosphate-buffered saline (PBS) or 10 9 vp of the indicated SC-Ads. Six weeks later, samples were collected for enzyme-linked immunosorbent assay (ELISA) vs. clade C CN54 gp140. ( A ) Sub-isotyping ELISA for the indicated samples at 1/200 dilution (low dilution used for low-sensitivity sub-isotyping kit). All IgG isotypes in the granulocyte macrophage colony-stimulating factor (GMCSF) and TcdA/B groups were significantly different than PBS by 2-way ANOVA. ( B ) ELISA OD450 levels are shown for 1/35 dilution of vaginal wash samples with detection by anti-IgA. * p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    IM or IN SOSIP protein boost of SC-Ad-env + SC-Ad-genetic adjuvants. The groups of 10 mice from Figure 3 were divided and boosted with 5 µg CZA clade C SOSIP protein adjuvanted with 1 µg alphaGalCer by either the IM or IN route. Two weeks later, 1/35 dilutions of vaginal washes were assay for anti-CN54 IgG or IgA antibodies by ELISA.
    Figure Legend Snippet: IM or IN SOSIP protein boost of SC-Ad-env + SC-Ad-genetic adjuvants. The groups of 10 mice from Figure 3 were divided and boosted with 5 µg CZA clade C SOSIP protein adjuvanted with 1 µg alphaGalCer by either the IM or IN route. Two weeks later, 1/35 dilutions of vaginal washes were assay for anti-CN54 IgG or IgA antibodies by ELISA.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    15) Product Images from "The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor"

    Article Title: The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.18.12081-12087.2005

    Colocalization of bikunin with ORF3 in liver cells. Huh7 cells transiently transfected with pMT-ORF3 were doubly labeled with polyclonal antibikunin and monoclonal anti-ORF3, followed by the Alexa 488 or Alexa 594 conjugated anti-rabbit IgG or anti-mouse
    Figure Legend Snippet: Colocalization of bikunin with ORF3 in liver cells. Huh7 cells transiently transfected with pMT-ORF3 were doubly labeled with polyclonal antibikunin and monoclonal anti-ORF3, followed by the Alexa 488 or Alexa 594 conjugated anti-rabbit IgG or anti-mouse

    Techniques Used: Transfection, Labeling

    16) Product Images from "A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane"

    Article Title: A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

    Journal: The EMBO Journal

    doi: 10.1093/emboj/21.11.2557

    Fig. 5. Co-localization and functional interaction of EHD1 with Arf6. HeLa cells were transiently co-transfected with constructs encoding Myc-EHD1 and wild-type Arf6 ( A – C ), GFP–EHD1 and Arf6-Q67L ( D – F ), GFP–EHD1 and Arf6-T27N ( G – I ), GFP–EHD1 and FLAG-EFA6 ( J – L ), and GFP–EHD1 and FLAG-ACAP1 ( M – O ). Cells were fixed, permeabilized and incubated with a monoclonal antibody to the Myc epitope and a rabbit polyclonal antibody to Arf6 (A–C). Bound antibodies were revealed by Alexa-488-conjugated antibody to mouse IgG (A and C), and by Cy3-conjugated anti-rabbit IgG (B and C). HeLa cells co-transfected with GFP–EHD1 and Arf6 mutant constructs (D–I) were fixed, permeabilized and incubated with a rabbit polyclonal antibody directed against Arf6 (D–I), followed by Cy3-conjugated anti-rabbit IgG (E, F, H and I). HeLa cells co-transfected with GFP–EHD1 and FLAG-EFA6 (J–L) or FLAG-ACAP1 (M–O) were fixed, permeabilized and incubated with a monoclonal antibody to the FLAG epitope, followed by a Cy3-conjugated anti-mouse IgG antibody. All images were obtained by confocal microscopy. Arrows (A and B) denote tubular structures containing both Arf6 and EHD1. Bar, 10 µm.
    Figure Legend Snippet: Fig. 5. Co-localization and functional interaction of EHD1 with Arf6. HeLa cells were transiently co-transfected with constructs encoding Myc-EHD1 and wild-type Arf6 ( A – C ), GFP–EHD1 and Arf6-Q67L ( D – F ), GFP–EHD1 and Arf6-T27N ( G – I ), GFP–EHD1 and FLAG-EFA6 ( J – L ), and GFP–EHD1 and FLAG-ACAP1 ( M – O ). Cells were fixed, permeabilized and incubated with a monoclonal antibody to the Myc epitope and a rabbit polyclonal antibody to Arf6 (A–C). Bound antibodies were revealed by Alexa-488-conjugated antibody to mouse IgG (A and C), and by Cy3-conjugated anti-rabbit IgG (B and C). HeLa cells co-transfected with GFP–EHD1 and Arf6 mutant constructs (D–I) were fixed, permeabilized and incubated with a rabbit polyclonal antibody directed against Arf6 (D–I), followed by Cy3-conjugated anti-rabbit IgG (E, F, H and I). HeLa cells co-transfected with GFP–EHD1 and FLAG-EFA6 (J–L) or FLAG-ACAP1 (M–O) were fixed, permeabilized and incubated with a monoclonal antibody to the FLAG epitope, followed by a Cy3-conjugated anti-mouse IgG antibody. All images were obtained by confocal microscopy. Arrows (A and B) denote tubular structures containing both Arf6 and EHD1. Bar, 10 µm.

    Techniques Used: Functional Assay, Transfection, Construct, Incubation, Mutagenesis, FLAG-tag, Confocal Microscopy

    Fig. 7. EHD1 tubular structures promote recycling of MHC-I to the cell surface. ( A ) Time-dependent co-localization of internalized MHC-I with EHD1 tubules by live image analysis. MHC-I monoclonal antibodies were coupled to Alexa Fluor 568 F(ab′)2 fragment of goat anti-mouse IgG. The coupled antibodies were then used to continuously pulse HeLa cells that were transfected 24 h earlier with a GFP–EHD1 construct. Images of MHC-I uptake (left panels) and GFP–EHD1 tubules (right panels) are depicted. Arrows (white) mark MHC-I tubular structures that appear at 15–20 min of internalization and co-localize with pre-existing GFP–EHD1 tubules (black arrows). Images are shown inverted to facilitate analysis (see Supplementary time-lapse video). Bar, 10 µm. ( B ) Quantification of EHD1-enhanced MHC-I recycling by a CELISA assay. HeLa cells were transfected with cDNA coding for H-2D d (mouse MHC-I), H-2D d and GFP–EHD1, H-2D d and Myc-EHD1, H-2D d and GFP–EHD1-G65R, or H-2D d and GFP–EHD1-K220N. Internalization of MHC-I over time was monitored 24 h after transfection by CELISA utilizing a biotinylated anti-MHC-I antibody (see Materials and methods), and the fraction of MHC-I antibody on the surface at each time point was recorded. A representative experiment from four independent CELISA assays is depicted, with triplicates at each time point.
    Figure Legend Snippet: Fig. 7. EHD1 tubular structures promote recycling of MHC-I to the cell surface. ( A ) Time-dependent co-localization of internalized MHC-I with EHD1 tubules by live image analysis. MHC-I monoclonal antibodies were coupled to Alexa Fluor 568 F(ab′)2 fragment of goat anti-mouse IgG. The coupled antibodies were then used to continuously pulse HeLa cells that were transfected 24 h earlier with a GFP–EHD1 construct. Images of MHC-I uptake (left panels) and GFP–EHD1 tubules (right panels) are depicted. Arrows (white) mark MHC-I tubular structures that appear at 15–20 min of internalization and co-localize with pre-existing GFP–EHD1 tubules (black arrows). Images are shown inverted to facilitate analysis (see Supplementary time-lapse video). Bar, 10 µm. ( B ) Quantification of EHD1-enhanced MHC-I recycling by a CELISA assay. HeLa cells were transfected with cDNA coding for H-2D d (mouse MHC-I), H-2D d and GFP–EHD1, H-2D d and Myc-EHD1, H-2D d and GFP–EHD1-G65R, or H-2D d and GFP–EHD1-K220N. Internalization of MHC-I over time was monitored 24 h after transfection by CELISA utilizing a biotinylated anti-MHC-I antibody (see Materials and methods), and the fraction of MHC-I antibody on the surface at each time point was recorded. A representative experiment from four independent CELISA assays is depicted, with triplicates at each time point.

    Techniques Used: Transfection, Construct

    17) Product Images from "Presence of a neo-epitope and absence of amyloid beta and tau protein in degenerative hippocampal granules of aged mice"

    Article Title: Presence of a neo-epitope and absence of amyloid beta and tau protein in degenerative hippocampal granules of aged mice

    Journal: Age

    doi: 10.1007/s11357-013-9560-9

    Immunohistochemical staining of the brain sections from SAMP8 mice with Tau5A antibody. a Secondary antibody against IgG. b Secondary antibody against IgM. c Merging of a and b. Yellow corresponds to colocalisation of both stainings. Scale bar 100 μm
    Figure Legend Snippet: Immunohistochemical staining of the brain sections from SAMP8 mice with Tau5A antibody. a Secondary antibody against IgG. b Secondary antibody against IgM. c Merging of a and b. Yellow corresponds to colocalisation of both stainings. Scale bar 100 μm

    Techniques Used: Immunohistochemistry, Staining, Mouse Assay

    18) Product Images from "A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G > A (IVS25+1G > A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor"

    Article Title: A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G > A (IVS25+1G > A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059537

    Cell surface expression and shedding of mutant ACE. Panel A. FACS analysis of immature dendritic cells with anti-ACE antibodies. Cells were stained with mAb i2H5 (black histogram) or with control mouse IgG (grey histogram). Figures correspond to values of median fluorescence intensity. Affected family members with the IVS25+1G > A mutation were shown to express a ≈50% lower level of ACE on their cell surface compared to non-affected family members. Panel B. ACE shedding into the medium. ACE activity in the medium (“Soluble ACE”) and membrane-bound ACE activity were determined on immature dendritic cells from 2 subjects harboring the IVS25+1G > A mutation (II.7 and III.1) and 2 controls (II.6 and I.9) from the first pedigree. Their ratio represents the rate of ACE shedding into the medium. In agreement with the findings of the FACS analysis, shedding was ≈3 times larger in the individuals with the mutated ACE. WT: wild-type subjects.
    Figure Legend Snippet: Cell surface expression and shedding of mutant ACE. Panel A. FACS analysis of immature dendritic cells with anti-ACE antibodies. Cells were stained with mAb i2H5 (black histogram) or with control mouse IgG (grey histogram). Figures correspond to values of median fluorescence intensity. Affected family members with the IVS25+1G > A mutation were shown to express a ≈50% lower level of ACE on their cell surface compared to non-affected family members. Panel B. ACE shedding into the medium. ACE activity in the medium (“Soluble ACE”) and membrane-bound ACE activity were determined on immature dendritic cells from 2 subjects harboring the IVS25+1G > A mutation (II.7 and III.1) and 2 controls (II.6 and I.9) from the first pedigree. Their ratio represents the rate of ACE shedding into the medium. In agreement with the findings of the FACS analysis, shedding was ≈3 times larger in the individuals with the mutated ACE. WT: wild-type subjects.

    Techniques Used: Expressing, Mutagenesis, FACS, Staining, Fluorescence, Activity Assay

    19) Product Images from "Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations"

    Article Title: Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations

    Journal: Scientific Reports

    doi: 10.1038/srep16279

    Correlating 3D-SIM and d STORM images of a rat liver sinusoidal endothelial cell (LSEC). ( A ) Maximum intensity z-projection 3D-SIM image of a 4-color-stained fixed rat LSEC. The nucleus was stained with DAPI (blue), actin filaments with Phalloidin-Alexa488 (green), membranes with CellMask Orange (white), and tubulin structures with anti β-tubulin mouse antibody followed by an anti-mouse IgG-Alexa647 antibody (magenta). The maximum intensity z-projection corresponds to a sample thickness of 750 nm. ( B ) Maximum intensity z-projection 3D-SIM image of the tubulin channel from (A) compared to the d STORM reconstruction ( C ) of the same cell. ( D ) Enlarged 3D-SIM and ( E ) d STORM images of the ROIs (dashed-line boxes) shown in ( B,C ). The d STORM image shows a direct correlation with the corresponding 3D-SIM image, but with an optical resolution of approx. 20 nm. Note that the d STORM image is obtained in HiLo mode, where in thicker parts of the cell not all of the entire volume of the cell is illuminated, resulting in small differences between the images. The single frame exposure time of the d STORM image was 20 ms and a total of 10000 frames were used for the reconstruction. The sample was mounted in Vectashield.
    Figure Legend Snippet: Correlating 3D-SIM and d STORM images of a rat liver sinusoidal endothelial cell (LSEC). ( A ) Maximum intensity z-projection 3D-SIM image of a 4-color-stained fixed rat LSEC. The nucleus was stained with DAPI (blue), actin filaments with Phalloidin-Alexa488 (green), membranes with CellMask Orange (white), and tubulin structures with anti β-tubulin mouse antibody followed by an anti-mouse IgG-Alexa647 antibody (magenta). The maximum intensity z-projection corresponds to a sample thickness of 750 nm. ( B ) Maximum intensity z-projection 3D-SIM image of the tubulin channel from (A) compared to the d STORM reconstruction ( C ) of the same cell. ( D ) Enlarged 3D-SIM and ( E ) d STORM images of the ROIs (dashed-line boxes) shown in ( B,C ). The d STORM image shows a direct correlation with the corresponding 3D-SIM image, but with an optical resolution of approx. 20 nm. Note that the d STORM image is obtained in HiLo mode, where in thicker parts of the cell not all of the entire volume of the cell is illuminated, resulting in small differences between the images. The single frame exposure time of the d STORM image was 20 ms and a total of 10000 frames were used for the reconstruction. The sample was mounted in Vectashield.

    Techniques Used: Staining, Mass Spectrometry

    20) Product Images from "The lupus susceptibility locus Sle1 breaches peripheral B cell tolerance at the antibody forming cell and germinal center checkpoints 1"

    Article Title: The lupus susceptibility locus Sle1 breaches peripheral B cell tolerance at the antibody forming cell and germinal center checkpoints 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0804215

    Elevated bone marrow long-lived IgM and IgG AFC responses from canonical HKIR cells bearing Sle1 MACS-purified HKIR and HKIR. Sle1 total (2× 10 6 ) B cells were transferred into B6 mice, the mice were immunized with Ars-KLH and the number of E4 + IgM (A, top) and E4 + IgG (B, bottom) AFCs were measured by ELISpot 30 days post-transfer/immunization. Each circle represents data from an individual chimeric mouse. Horizontal bars represent the average number of E4 + AFCs in each data set. All statistical analyses were performed by Student’s t-test.
    Figure Legend Snippet: Elevated bone marrow long-lived IgM and IgG AFC responses from canonical HKIR cells bearing Sle1 MACS-purified HKIR and HKIR. Sle1 total (2× 10 6 ) B cells were transferred into B6 mice, the mice were immunized with Ars-KLH and the number of E4 + IgM (A, top) and E4 + IgG (B, bottom) AFCs were measured by ELISpot 30 days post-transfer/immunization. Each circle represents data from an individual chimeric mouse. Horizontal bars represent the average number of E4 + AFCs in each data set. All statistical analyses were performed by Student’s t-test.

    Techniques Used: Magnetic Cell Separation, Purification, Mouse Assay, Enzyme-linked Immunospot

    Primary IgM and IgG AFC responses of HKIR canonical B cells in the presence or absence of Sle1 HKIR and HKIR. Sle1 canonical B cells were transferred to B6 recipients that were subsequently immunized with Ars-KLH (see Materials and Methods ). The number of splenic E4 + IgM (A, upper panel) and IgG (B, lower panel) secreting AFCs were measured by ELISpot assay 6 days after immunization. Each circle represents the number of E4 + AFCs per 1×10 6 splenocytes obtained from an individual chimeric mouse. Open and closed circles represent data from HKIR and HKIR. Sle1 chimeric mice, respectively. Horizontal bars represent the average number of E4 + AFCs. Statistical analysis was performed by Student’s t-test. The data for IgG producing AFCs were obtained from three independent experiments and the data for IgM producing AFCs were obtained from two independent experiments.
    Figure Legend Snippet: Primary IgM and IgG AFC responses of HKIR canonical B cells in the presence or absence of Sle1 HKIR and HKIR. Sle1 canonical B cells were transferred to B6 recipients that were subsequently immunized with Ars-KLH (see Materials and Methods ). The number of splenic E4 + IgM (A, upper panel) and IgG (B, lower panel) secreting AFCs were measured by ELISpot assay 6 days after immunization. Each circle represents the number of E4 + AFCs per 1×10 6 splenocytes obtained from an individual chimeric mouse. Open and closed circles represent data from HKIR and HKIR. Sle1 chimeric mice, respectively. Horizontal bars represent the average number of E4 + AFCs. Statistical analysis was performed by Student’s t-test. The data for IgG producing AFCs were obtained from three independent experiments and the data for IgM producing AFCs were obtained from two independent experiments.

    Techniques Used: Enzyme-linked Immunospot, Mouse Assay

    21) Product Images from "Mechanisms of Assembly and Cellular Interactions for the Bacterial Genotoxin CDT"

    Article Title: Mechanisms of Assembly and Cellular Interactions for the Bacterial Genotoxin CDT

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0010028

    The Activity of CDT Holotoxin Labeled with Alexa Fluor 488 (A) The toxicity of CDT holotoxin labeled with Alexa Fluor 488. HeLa cells were treated with 1 ng/ml (black) or 10 ng/ml (gray) concentration of unconjugated or Alexa Fluor 488−conjugated CDT for 3 hr at 37°C, 5% CO 2 . Cells were processed 48 hr after holotoxin treatment, and DNA content was measured by flow cytometry. The calculated percentages of cells in G0/G1, S, and G2/M are shown. (B) Binding of CDT-Alexa Fluor 488 to cells. Harvested HeLa cells were exposed for 2 hr to 5 and 10 μg/ml concentration of wild-type or mutant CDT-Alexa Fluor 488. The histogram shows the binding of 5 or 10 μg/ml concentration of wild-type and mutant CDT-Alexa Fluor 488 conjugates to HeLa cells. Mock represents cells in buffer only (2% FCS in PBS), and control is goat anti-mouse IgG conjugated with Alexa Fluor 488, which does not bind to HeLa cells. The level of fluorescence was analyzed by flow cytometry. The relative levels of fluorescent labeling of wild-type and mutant CDT holotoxin was maintained to be nearly equivalent, with the mutant holotoxins (groove and aromatic patch) possessing a slightly higher level of labeling than the wild-type (Materials and Methods).
    Figure Legend Snippet: The Activity of CDT Holotoxin Labeled with Alexa Fluor 488 (A) The toxicity of CDT holotoxin labeled with Alexa Fluor 488. HeLa cells were treated with 1 ng/ml (black) or 10 ng/ml (gray) concentration of unconjugated or Alexa Fluor 488−conjugated CDT for 3 hr at 37°C, 5% CO 2 . Cells were processed 48 hr after holotoxin treatment, and DNA content was measured by flow cytometry. The calculated percentages of cells in G0/G1, S, and G2/M are shown. (B) Binding of CDT-Alexa Fluor 488 to cells. Harvested HeLa cells were exposed for 2 hr to 5 and 10 μg/ml concentration of wild-type or mutant CDT-Alexa Fluor 488. The histogram shows the binding of 5 or 10 μg/ml concentration of wild-type and mutant CDT-Alexa Fluor 488 conjugates to HeLa cells. Mock represents cells in buffer only (2% FCS in PBS), and control is goat anti-mouse IgG conjugated with Alexa Fluor 488, which does not bind to HeLa cells. The level of fluorescence was analyzed by flow cytometry. The relative levels of fluorescent labeling of wild-type and mutant CDT holotoxin was maintained to be nearly equivalent, with the mutant holotoxins (groove and aromatic patch) possessing a slightly higher level of labeling than the wild-type (Materials and Methods).

    Techniques Used: Activity Assay, Labeling, Concentration Assay, Flow Cytometry, Cytometry, Binding Assay, Mutagenesis, Fluorescence

    22) Product Images from "B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection"

    Article Title: B Cell-Specific Expression of Ataxia-Telangiectasia Mutated Protein Kinase Promotes Chronic Gammaherpesvirus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.01103-17

    ATM deficiency in B cells attenuates MHV68-driven B cell differentiation. B-Cre-positive and -negative mice were either mock treated or infected intranasally with 10 4 PFU of MHV68. At 16 days postinfection (dpi), splenocytes were harvested and analyzed using flow cytometry. Each data point represents an individual mouse; data from 2 to 4 independent experiments were pooled. (A) Class-switched B cells were pregated on B220 + and identified as IgM − IgD − (a representative flow diagram is shown). Boxed areas identify immune populations of interest. (B and C) The frequencies (B) and the absolute numbers (C) of B220 + IgM − IgD − splenocytes were quantified. (D) Germinal center B cells were pregated on B220 + and further identified as CD95 + GL7 + (a representative flow diagram is shown). (E and F) Frequencies (E) and absolute numbers (F) of B220 + CD95 + GL7 + splenocytes. (G) Plasma cells were pregated on B220 + , further gated as IgM − IgD − , and identified for surface expression of CD138 + and for intracellular IgG + (representative flow diagrams are shown). (H and I) Frequencies (H) and absolute numbers (I) of B220 + IgM − IgD − CD138 + IgG + plasma cells. (J to M) Serum total immunoglobulin (J) and MHV68-specific antibodies (total [K], IgM [L], and IgG [M]) were measured by ELISA at 16 days post-mock treatment or -MHV68 infection; pooled data are shown. *, P
    Figure Legend Snippet: ATM deficiency in B cells attenuates MHV68-driven B cell differentiation. B-Cre-positive and -negative mice were either mock treated or infected intranasally with 10 4 PFU of MHV68. At 16 days postinfection (dpi), splenocytes were harvested and analyzed using flow cytometry. Each data point represents an individual mouse; data from 2 to 4 independent experiments were pooled. (A) Class-switched B cells were pregated on B220 + and identified as IgM − IgD − (a representative flow diagram is shown). Boxed areas identify immune populations of interest. (B and C) The frequencies (B) and the absolute numbers (C) of B220 + IgM − IgD − splenocytes were quantified. (D) Germinal center B cells were pregated on B220 + and further identified as CD95 + GL7 + (a representative flow diagram is shown). (E and F) Frequencies (E) and absolute numbers (F) of B220 + CD95 + GL7 + splenocytes. (G) Plasma cells were pregated on B220 + , further gated as IgM − IgD − , and identified for surface expression of CD138 + and for intracellular IgG + (representative flow diagrams are shown). (H and I) Frequencies (H) and absolute numbers (I) of B220 + IgM − IgD − CD138 + IgG + plasma cells. (J to M) Serum total immunoglobulin (J) and MHV68-specific antibodies (total [K], IgM [L], and IgG [M]) were measured by ELISA at 16 days post-mock treatment or -MHV68 infection; pooled data are shown. *, P

    Techniques Used: Cell Differentiation, Mouse Assay, Infection, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

    23) Product Images from "RBM39 alters phosphorylation of c-Jun and binds to viral RNA to promote PRRSV proliferation"

    Article Title: RBM39 alters phosphorylation of c-Jun and binds to viral RNA to promote PRRSV proliferation

    Journal: bioRxiv

    doi: 10.1101/2020.11.13.382531

    Interaction and co-localization between RBM39 and c-Jun. (A) HEK 293T cells were co-transfected with Flag-c-Jun (or empty vector) and HA-RBM39. (B) The plasmids Flag-RBM39 (full-length), RRM2, RRM3, ΔRRM1, ΔRRM or empty vector and pHA-c-Jun was co-transfected into HEK293T respectively. At 24 h after transfection, the cells lysates of (A) and (B) were precipitated with anti-Flag-labeled beads (Sigma) and were further detected by WB with anti-Flag/HA antibody. (C-F) HEK293T cell were co-transfected with pFlag-RBM39+pHA-c-Jun or pFlag-c-Jun+pHA-RBM39. After 24 h, cells were fixed and doubly stained with rabbit anti-HA mAb and mouse anti-Flag antibody followed by FITC-conjugated anti-rabbit IgG (green) or IF555-conjugated anti-mouse IgG (red). Nuclei were stained with Hoechst 33258 dye (blue). Interaction and nuclear localization of RBM39 and c-Jun were observed using Laser confocal fluorescence microscope. Scale bar: 14μm. Data are representative of results from three independent experiments.
    Figure Legend Snippet: Interaction and co-localization between RBM39 and c-Jun. (A) HEK 293T cells were co-transfected with Flag-c-Jun (or empty vector) and HA-RBM39. (B) The plasmids Flag-RBM39 (full-length), RRM2, RRM3, ΔRRM1, ΔRRM or empty vector and pHA-c-Jun was co-transfected into HEK293T respectively. At 24 h after transfection, the cells lysates of (A) and (B) were precipitated with anti-Flag-labeled beads (Sigma) and were further detected by WB with anti-Flag/HA antibody. (C-F) HEK293T cell were co-transfected with pFlag-RBM39+pHA-c-Jun or pFlag-c-Jun+pHA-RBM39. After 24 h, cells were fixed and doubly stained with rabbit anti-HA mAb and mouse anti-Flag antibody followed by FITC-conjugated anti-rabbit IgG (green) or IF555-conjugated anti-mouse IgG (red). Nuclei were stained with Hoechst 33258 dye (blue). Interaction and nuclear localization of RBM39 and c-Jun were observed using Laser confocal fluorescence microscope. Scale bar: 14μm. Data are representative of results from three independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Labeling, Western Blot, Staining, Fluorescence, Microscopy

    RBM39 down-regulates AP-1 signaling pathway and PRRSV infection triggers out of the nucleus of translocation of RBM39 and c-Jun. (A and B) HEK293 T cells were transfected or co-transfected with AP-1 promoter reporter plasmid and different concentrations of HA-RBM39. The luciferase activity of AP-1 promoter reporter gene was measured through Dual Luciferase Reporter Gene Assay Kit (Yeasen Technology). (C, D and F)The 3D4/21cells were respectively transfected or co-transfected with HA-RBM39 and Flag-c-Jun plasmids, 12 h after transfection, cells were infected with PRRSV at an MOI of 0.4. After 24 h, cells were fixed and doubly stained with rabbit anti-HA mAb and mouse anti-Flag antibody followed by FITC-conjugated anti-rabbit IgG (green) or IF555-conjugated anti-mouse IgG (red). Nuclei were stained with Hoechst 33258 dye (blue). Interaction and nuclear localization of RBM39 and c-Jun were observed using Laser confocal fluorescence microscope. Scale bar: 14μm. * P
    Figure Legend Snippet: RBM39 down-regulates AP-1 signaling pathway and PRRSV infection triggers out of the nucleus of translocation of RBM39 and c-Jun. (A and B) HEK293 T cells were transfected or co-transfected with AP-1 promoter reporter plasmid and different concentrations of HA-RBM39. The luciferase activity of AP-1 promoter reporter gene was measured through Dual Luciferase Reporter Gene Assay Kit (Yeasen Technology). (C, D and F)The 3D4/21cells were respectively transfected or co-transfected with HA-RBM39 and Flag-c-Jun plasmids, 12 h after transfection, cells were infected with PRRSV at an MOI of 0.4. After 24 h, cells were fixed and doubly stained with rabbit anti-HA mAb and mouse anti-Flag antibody followed by FITC-conjugated anti-rabbit IgG (green) or IF555-conjugated anti-mouse IgG (red). Nuclei were stained with Hoechst 33258 dye (blue). Interaction and nuclear localization of RBM39 and c-Jun were observed using Laser confocal fluorescence microscope. Scale bar: 14μm. * P

    Techniques Used: Infection, Translocation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Gene Assay, Staining, Fluorescence, Microscopy

    24) Product Images from "Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA"

    Article Title: Cross-Species Immune Recognition Between Plasmodium vivax Duffy Binding Protein Antibodies and the Plasmodium falciparum Surface Antigen VAR2CSA

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiy467

    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells (RBCs) to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. IgG1, immunoglobulin G1.
    Figure Legend Snippet: Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 blocks adhesion of infected red blood cells (RBCs) to chondroitin sulfate A (CSA). A , Controls for the inhibition of binding assay included Plasmodium falciparum strain–CS2 infected RBCs incubated with phosphate-buffered saline (PBS) alone, soluble CSA (sCSA), and sera from primigravid and multigravid women from Uganda. B – D , PvDBP mAb 3D10 was tested for inhibition of CS2 ( B ), a placental isolate ( C ), and NF54-CSA–infected RBC binding to CSA ( D ). Results are expressed as the number of parasites bound to CSA from replicates of a representative experiment. IgG1, immunoglobulin G1.

    Techniques Used: Binding Assay, Infection, Inhibition, Incubation

    Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 recognizes live Plasmodium falciparum strain CS2–infected red blood cells (RBCs). CS2, a placental isolate, and NF54–chondroitin sulfate A (CSA) infected RBCs were analyzed by flow cytometry. A and B , To verify the expression of VAR2CSA, infected RBCs were stained with normal rabbit serum ( A ) and a polyclonal anti-VAR2CSA rabbit antibody ( B ), both at a 1:40 dilution. C and D , All 3 strains were stained with the immunoglobulin G1 (IgG1) isotype control ( C ) and PvDBP 3D10 mAb ( D ), both at 143 μg/mL. The percentage of infected RBCs recognized by the antibody is indicated on each plot.
    Figure Legend Snippet: Plasmodium vivax Duffy binding protein (PvDBP) monoclonal antibody (mAb) 3D10 recognizes live Plasmodium falciparum strain CS2–infected red blood cells (RBCs). CS2, a placental isolate, and NF54–chondroitin sulfate A (CSA) infected RBCs were analyzed by flow cytometry. A and B , To verify the expression of VAR2CSA, infected RBCs were stained with normal rabbit serum ( A ) and a polyclonal anti-VAR2CSA rabbit antibody ( B ), both at a 1:40 dilution. C and D , All 3 strains were stained with the immunoglobulin G1 (IgG1) isotype control ( C ) and PvDBP 3D10 mAb ( D ), both at 143 μg/mL. The percentage of infected RBCs recognized by the antibody is indicated on each plot.

    Techniques Used: Binding Assay, Infection, Flow Cytometry, Cytometry, Expressing, Staining

    25) Product Images from "Intake of Lactobacillus delbrueckii (pExu:hsp65) Prevents the Inflammation and the Disorganization of the Intestinal Mucosa in a Mouse Model of Mucositis"

    Article Title: Intake of Lactobacillus delbrueckii (pExu:hsp65) Prevents the Inflammation and the Disorganization of the Intestinal Mucosa in a Mouse Model of Mucositis

    Journal: Microorganisms

    doi: 10.3390/microorganisms9010107

    Hsp65 expression in transfected eukaryotic cells. Confocal microscopy: ( A ) negative control: non-transfected Chinese Hamster Ovarian cell line (CHO) cells; ( B ) negative control: non-transfected eukaryotic cells labeled with primary (Mab anti-Hsp65) and secondary (goat anti-mouse IgG (H + L)) antibodies labeled with Alexa 488. ( C , D ) CHO cells labeled with primary (Mb_HSP65) and secondary (goat anti-mouse IgG (H + L)) antibodies. In green, the Hsp65 protein is expressed in the cytoplasm of eukaryotic cells. 2D images ( A – D ) were acquired in both depths (z-stack) using a Zeiss LSM 510 META inverted confocal laser 1358 scanning microscope with 40× or 60× objective.
    Figure Legend Snippet: Hsp65 expression in transfected eukaryotic cells. Confocal microscopy: ( A ) negative control: non-transfected Chinese Hamster Ovarian cell line (CHO) cells; ( B ) negative control: non-transfected eukaryotic cells labeled with primary (Mab anti-Hsp65) and secondary (goat anti-mouse IgG (H + L)) antibodies labeled with Alexa 488. ( C , D ) CHO cells labeled with primary (Mb_HSP65) and secondary (goat anti-mouse IgG (H + L)) antibodies. In green, the Hsp65 protein is expressed in the cytoplasm of eukaryotic cells. 2D images ( A – D ) were acquired in both depths (z-stack) using a Zeiss LSM 510 META inverted confocal laser 1358 scanning microscope with 40× or 60× objective.

    Techniques Used: Expressing, Transfection, Confocal Microscopy, Negative Control, Labeling, Microscopy

    26) Product Images from "Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice"

    Article Title: Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124664

    Specific antibodies determined by ELISA. Sera samples were collected from mice immunized with rOmpB-4 combined with C . burnetii CMR (O+CMR-C), C . burnetii CMR alone (CMR-C), or rOmpB-4 alone (rOmpB-4) on days 7, 14, 21, and 28 days after first immunization, respectively. IgG (A), IgG1 (B), or IgG2a (C) to rOmpB-4 in sera was determined by ELISA and the ratio of IgG2a/IgG1 in each serum sample was also compared (D). The statistically significant differences of OD 450 on 28 days after first immunization among groups were analyzed using the Student’s t- test or Wilcoxon two-sample test according to their normality and homogeneity of variance. Results were expressed as mean ± SD ( n = 3). P
    Figure Legend Snippet: Specific antibodies determined by ELISA. Sera samples were collected from mice immunized with rOmpB-4 combined with C . burnetii CMR (O+CMR-C), C . burnetii CMR alone (CMR-C), or rOmpB-4 alone (rOmpB-4) on days 7, 14, 21, and 28 days after first immunization, respectively. IgG (A), IgG1 (B), or IgG2a (C) to rOmpB-4 in sera was determined by ELISA and the ratio of IgG2a/IgG1 in each serum sample was also compared (D). The statistically significant differences of OD 450 on 28 days after first immunization among groups were analyzed using the Student’s t- test or Wilcoxon two-sample test according to their normality and homogeneity of variance. Results were expressed as mean ± SD ( n = 3). P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

    Specific antibodies determined by IFA. Serum samples were collected from mice immunized with rOmpB-4 combined C . burnetii CMR on days 7, 14, 21, and 28 after first immunization, respectively. Anti- C . burnetii phase I/II IgG titers was evaluated by IFA.
    Figure Legend Snippet: Specific antibodies determined by IFA. Serum samples were collected from mice immunized with rOmpB-4 combined C . burnetii CMR on days 7, 14, 21, and 28 after first immunization, respectively. Anti- C . burnetii phase I/II IgG titers was evaluated by IFA.

    Techniques Used: Immunofluorescence, Mouse Assay

    27) Product Images from "Regulation of nerve growth and patterning by cell surface protein disulphide isomerase"

    Article Title: Regulation of nerve growth and patterning by cell surface protein disulphide isomerase

    Journal: bioRxiv

    doi: 10.1101/838771

    csPDI mediates axon repulsion in vitro a, Collapse assays testing purified bovine PDI in liposomes at a range of concentrations; controls, phosphate-buffered saline (PBS) and untreated liposomes; histogram shows mean +s.e.m. b, Assays testing PDI and GSNO applied individually or concomitantly. c, Assays testing PACMA31 and PACMA56 on somite extracts (SE). d, Assays testing reducing agents at the concentrations indicated when applied either alone or together with PDI+GSNO. e, Assays testing GSH and L-homocysteine on SE-induced collapse. f, Assays testing myoglobin (20μM) on SE- and PDI+GSNO-induced collapse. g, Assays testing carboxy (C)-PTIO (20μM) on PDI+GSNO-induced collapse. h, Assays testing L-NAME and its control D-NAME on SE-induced collapse; calcimycin was used as a positive control. i, S-nitrosylated protein (iodo-TMT-labelled) in somites; protein samples (48μg) from somite cell-free extracts were fractionated on NuPAGE 4-12% Bis Tris gels as described in the Methods; lanes 1 2 are controls consisting of somite proteins only, with no detectable signal compared with lanes 3 4 where GSNO has been added; lane 3 is a control (treated with water) showing negligible iodoTMT labelling, and lane 4 (reduced with ascorbate to generate a new free thiol for labelling) shows increased label; lane 5 shows that addition of PDI (1μg/0.1ml reaction mixture) enhances labelling; lane 6 shows that 3mM GSH in the absence of GSNO and PDI does not generate a signal; lane 7 shows that 3μM GSH is insufficient to interfere with nitrosylation, concurring with the findings of Sliskovic et al. 37 . The coloured molecular weight markers on the blot are shown on the left (BLUeye prestained protein ladder, 2.5 μL, Geneflow). j, Identification of LC1 in somite extract (25μg protein); the blot was cut in half above the 41K marker and the top half of the membrane was probed with rabbit anti-tubulin followed by goat anti-rabbit IgG; the bottom half was probed with mouse monoclonal antibody against amino acids 2257-2357 of mouse MAP-1B (LC1) followed by goat anti-mouse IgG. The molecular weight markers on the blot are shown to the right (BLUeye prestained protein ladder, 3μl). k, Identification of LC1 as a substrate for S-nitrosylation; cell-free somite extract (200μg) was treated with D-NAME or L-NAME, followed by further incubation in GSNO (200μM), as described in the Methods. Samples were then processed for the presence of S-nitrosylated proteins using iodo-TMT as described in the Methods. Protein samples (15μg) were then fractionated and blotted, after which the blot was cut as described for Fig. 3j . The top half was treated with anti-tubulin and the bottom half with anti-iodoTMT. L-NAME treatment blocked S-nitrosylation, as shown by the lack of iodoTMT labelling. The control D-NAME was without effect.
    Figure Legend Snippet: csPDI mediates axon repulsion in vitro a, Collapse assays testing purified bovine PDI in liposomes at a range of concentrations; controls, phosphate-buffered saline (PBS) and untreated liposomes; histogram shows mean +s.e.m. b, Assays testing PDI and GSNO applied individually or concomitantly. c, Assays testing PACMA31 and PACMA56 on somite extracts (SE). d, Assays testing reducing agents at the concentrations indicated when applied either alone or together with PDI+GSNO. e, Assays testing GSH and L-homocysteine on SE-induced collapse. f, Assays testing myoglobin (20μM) on SE- and PDI+GSNO-induced collapse. g, Assays testing carboxy (C)-PTIO (20μM) on PDI+GSNO-induced collapse. h, Assays testing L-NAME and its control D-NAME on SE-induced collapse; calcimycin was used as a positive control. i, S-nitrosylated protein (iodo-TMT-labelled) in somites; protein samples (48μg) from somite cell-free extracts were fractionated on NuPAGE 4-12% Bis Tris gels as described in the Methods; lanes 1 2 are controls consisting of somite proteins only, with no detectable signal compared with lanes 3 4 where GSNO has been added; lane 3 is a control (treated with water) showing negligible iodoTMT labelling, and lane 4 (reduced with ascorbate to generate a new free thiol for labelling) shows increased label; lane 5 shows that addition of PDI (1μg/0.1ml reaction mixture) enhances labelling; lane 6 shows that 3mM GSH in the absence of GSNO and PDI does not generate a signal; lane 7 shows that 3μM GSH is insufficient to interfere with nitrosylation, concurring with the findings of Sliskovic et al. 37 . The coloured molecular weight markers on the blot are shown on the left (BLUeye prestained protein ladder, 2.5 μL, Geneflow). j, Identification of LC1 in somite extract (25μg protein); the blot was cut in half above the 41K marker and the top half of the membrane was probed with rabbit anti-tubulin followed by goat anti-rabbit IgG; the bottom half was probed with mouse monoclonal antibody against amino acids 2257-2357 of mouse MAP-1B (LC1) followed by goat anti-mouse IgG. The molecular weight markers on the blot are shown to the right (BLUeye prestained protein ladder, 3μl). k, Identification of LC1 as a substrate for S-nitrosylation; cell-free somite extract (200μg) was treated with D-NAME or L-NAME, followed by further incubation in GSNO (200μM), as described in the Methods. Samples were then processed for the presence of S-nitrosylated proteins using iodo-TMT as described in the Methods. Protein samples (15μg) were then fractionated and blotted, after which the blot was cut as described for Fig. 3j . The top half was treated with anti-tubulin and the bottom half with anti-iodoTMT. L-NAME treatment blocked S-nitrosylation, as shown by the lack of iodoTMT labelling. The control D-NAME was without effect.

    Techniques Used: In Vitro, Purification, Positive Control, Molecular Weight, Marker, Incubation

    28) Product Images from "MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts"

    Article Title: MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Extension formation does not require Rac Activity. NIH 3T3 cells were cotransfected with MSE55 and dominant-negative Rac. N17Rac transfected cells were detected with rabbit polyclonal anti-Myc antibody and goat anti-rabbit IgG-FITC (green). N17Rac-expressing cells produce multiple thin filopodia ( a and b ). N17Rac, MSE55, and F-actin were detected by using triple immunofluorescence. Cells coexpressing N17Rac and MSE55 form multiple thin filopodia and additionally form a single, long cellular extension ( c and d ). MSE55 was detected with M2 anti-FLAG mouse mAb and goat anti-mouse IgG-Alexa 350 (data not shown). Expression of dominant-negative N17Rac (green) was detected with a rabbit polyclonal antibody against c-Myc and with anti-rabbit IgG-FITC. F-actin was detected with Texas Red-phalloidin (red). (Bar = 20 μm.)
    Figure Legend Snippet: Extension formation does not require Rac Activity. NIH 3T3 cells were cotransfected with MSE55 and dominant-negative Rac. N17Rac transfected cells were detected with rabbit polyclonal anti-Myc antibody and goat anti-rabbit IgG-FITC (green). N17Rac-expressing cells produce multiple thin filopodia ( a and b ). N17Rac, MSE55, and F-actin were detected by using triple immunofluorescence. Cells coexpressing N17Rac and MSE55 form multiple thin filopodia and additionally form a single, long cellular extension ( c and d ). MSE55 was detected with M2 anti-FLAG mouse mAb and goat anti-mouse IgG-Alexa 350 (data not shown). Expression of dominant-negative N17Rac (green) was detected with a rabbit polyclonal antibody against c-Myc and with anti-rabbit IgG-FITC. F-actin was detected with Texas Red-phalloidin (red). (Bar = 20 μm.)

    Techniques Used: Activity Assay, Dominant Negative Mutation, Transfection, Expressing, Immunofluorescence

    MSE55 localizes to lamellipodia in Cos-7 cells. FLAG-tagged MSE55 was transfected into Cos-7 cells. Expression of wild-type MSE55 ( a ) or a CRIB mutant, MSE55-D36A, P41A, H47A ( c ), was detected with the M2 anti-FLAG mouse mAb and goat anti-mouse IgG-FITC. Staining of F-actin filaments was visualized with Texas Red-phalloidin ( b and d ). Fluorescent micrographs show MSE55 staining in membrane ruffles 18 hr after transfection ( a and b ). MSE55-D36A, P41A, H47A-expressing cells ( c ) showed less staining in the ruffles and more stress fibers ( d ). (Bar = 20 μm.)
    Figure Legend Snippet: MSE55 localizes to lamellipodia in Cos-7 cells. FLAG-tagged MSE55 was transfected into Cos-7 cells. Expression of wild-type MSE55 ( a ) or a CRIB mutant, MSE55-D36A, P41A, H47A ( c ), was detected with the M2 anti-FLAG mouse mAb and goat anti-mouse IgG-FITC. Staining of F-actin filaments was visualized with Texas Red-phalloidin ( b and d ). Fluorescent micrographs show MSE55 staining in membrane ruffles 18 hr after transfection ( a and b ). MSE55-D36A, P41A, H47A-expressing cells ( c ) showed less staining in the ruffles and more stress fibers ( d ). (Bar = 20 μm.)

    Techniques Used: Transfection, Expressing, Mutagenesis, Staining

    MSE55 induces long cellular extensions in NIH 3T3 cells. FLAG-tagged MSE55 was transfected into NIH 3T3 cells. Expression of wild-type MSE55 ( a ) or the MSE55-D36A, P41A, H47A CRIB mutant ( c ) was detected with M2 anti-FLAG mouse mAb and goat anti-mouse IgG-FITC. Staining of F-actin filaments also was visualized with Texas Red-phalloidin ( b and d ). Fluorescent micrographs show MSE55-expressing cells showing long cellular extensions ( a ) that colocalize with F-actin staining ( b ). Expression of MSE55-D36A, P41A, H47A did not show long cellular extensions ( c and d ). (Bar = 20 μm.) NIH 3T3 cells with extensions greater than 10 μm in length were counted in untransfected and both wild-type and mutant MSE55-transfected cells ( e ). Results are expressed as the mean percentage of cells (±SD) from three separate experiments.
    Figure Legend Snippet: MSE55 induces long cellular extensions in NIH 3T3 cells. FLAG-tagged MSE55 was transfected into NIH 3T3 cells. Expression of wild-type MSE55 ( a ) or the MSE55-D36A, P41A, H47A CRIB mutant ( c ) was detected with M2 anti-FLAG mouse mAb and goat anti-mouse IgG-FITC. Staining of F-actin filaments also was visualized with Texas Red-phalloidin ( b and d ). Fluorescent micrographs show MSE55-expressing cells showing long cellular extensions ( a ) that colocalize with F-actin staining ( b ). Expression of MSE55-D36A, P41A, H47A did not show long cellular extensions ( c and d ). (Bar = 20 μm.) NIH 3T3 cells with extensions greater than 10 μm in length were counted in untransfected and both wild-type and mutant MSE55-transfected cells ( e ). Results are expressed as the mean percentage of cells (±SD) from three separate experiments.

    Techniques Used: Transfection, Expressing, Mutagenesis, Staining

    29) Product Images from "Construction of Recombinant HVT Expressing PmpD, and Immunological Evaluation against Chlamydia psittaci and Marek’s Disease Virus"

    Article Title: Construction of Recombinant HVT Expressing PmpD, and Immunological Evaluation against Chlamydia psittaci and Marek’s Disease Virus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124992

    Confirmation of PmpD-N protein expression in HVT vector by immunoblotting assay and indirect immunofluorescence. (A) The PmpD-N expression in rHVT- pmpD -N was detected by immunoblot with C . psittaci strain 6BC-specific polyclonal antibodies. Lane M, pre-stained protein ladder; Lane 1, cell lysate post inoculation with rHVT- pmpD -N; Lane 2, cell lysate post inoculation with parental HVT. The black arrow indicates the approximately size of 43kDa. (B) Indirect immunofluorescence analysis of PmpD-N expression in CEF cells. CEF cells on glass coverslips were infected with rHVT- pmpD -N, then incubated with mouse anti-PmpD-N polyclonal antibody of C . psittaci and chicken anti-HVT polyclonal serum, and then reacted with goat anti-mouse IgG conjugated with Alexa Fluor 488 (green fluorescence, shown in the lower left panel) and goat anti-chicken IgY labelled with Alexa Fluor 568 (red fluorescence, shown in the lower right panel), respectively. Finally, cell nuclei were stained with DAPI (blue fluorescence, shown in the top right panel). The merged image is shown in the top left panel. The expression of the targeted protein is indicated by white arrows in top left panel. (C) Parental HVT control. CEF cells on glass coverslips were infected with parental HVT, and then the process of test and the panel meaning are the same as those shown in Fig 1B.
    Figure Legend Snippet: Confirmation of PmpD-N protein expression in HVT vector by immunoblotting assay and indirect immunofluorescence. (A) The PmpD-N expression in rHVT- pmpD -N was detected by immunoblot with C . psittaci strain 6BC-specific polyclonal antibodies. Lane M, pre-stained protein ladder; Lane 1, cell lysate post inoculation with rHVT- pmpD -N; Lane 2, cell lysate post inoculation with parental HVT. The black arrow indicates the approximately size of 43kDa. (B) Indirect immunofluorescence analysis of PmpD-N expression in CEF cells. CEF cells on glass coverslips were infected with rHVT- pmpD -N, then incubated with mouse anti-PmpD-N polyclonal antibody of C . psittaci and chicken anti-HVT polyclonal serum, and then reacted with goat anti-mouse IgG conjugated with Alexa Fluor 488 (green fluorescence, shown in the lower left panel) and goat anti-chicken IgY labelled with Alexa Fluor 568 (red fluorescence, shown in the lower right panel), respectively. Finally, cell nuclei were stained with DAPI (blue fluorescence, shown in the top right panel). The merged image is shown in the top left panel. The expression of the targeted protein is indicated by white arrows in top left panel. (C) Parental HVT control. CEF cells on glass coverslips were infected with parental HVT, and then the process of test and the panel meaning are the same as those shown in Fig 1B.

    Techniques Used: Expressing, Plasmid Preparation, Immunofluorescence, Staining, Infection, Incubation, Fluorescence

    30) Product Images from "Single-Particle Detection of Transcription following Rotavirus Entry"

    Article Title: Single-Particle Detection of Transcription following Rotavirus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.00651-17

    NSP2 and Atto 565 oligonucleotide probe colocalization in TLP-infected cells. BSC-1 cells were infected at an MOI of 20 (top row) or mock infected (bottom row). Infection was allowed to proceed at 37°C for 6 h. After paraformaldehyde fixation, the cells were permeabilized with 1% Triton X-100 and probed with a primary, NSP2-specific antibody, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then incubated overnight with the pool of Atto 565-labeled oligonucleotides and imaged. Maximum-intensity z-projections of the 488-nm channel (left), the 561-nm channel (middle), and the overlay of the two channels (right) are shown.
    Figure Legend Snippet: NSP2 and Atto 565 oligonucleotide probe colocalization in TLP-infected cells. BSC-1 cells were infected at an MOI of 20 (top row) or mock infected (bottom row). Infection was allowed to proceed at 37°C for 6 h. After paraformaldehyde fixation, the cells were permeabilized with 1% Triton X-100 and probed with a primary, NSP2-specific antibody, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then incubated overnight with the pool of Atto 565-labeled oligonucleotides and imaged. Maximum-intensity z-projections of the 488-nm channel (left), the 561-nm channel (middle), and the overlay of the two channels (right) are shown.

    Techniques Used: Infection, Incubation, Labeling

    Viroplasm localization with respect to fluorescently labeled DLPs. (A) Cells were infected at an MOI of 5 for 10 min with doubly labeled rcTLPs (VP7, Atto 488; DLP, Atto 647N) and washed, and infection was allowed to continue for 5 h. Paraformaldehyde fixation was followed by overnight incubation with the pool of 44 Atto 565-labeled oligonucleotide probes and subsequent 3D imaging at 640-nm (left), 488-nm (middle), and 561-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (488-nm channel), colocalized with a strong signal in the Atto 565 oligonucleotide channel (561 nm). Green boxes, similarly large oligonucleotide bodies that do not colocalize with any DLP signal. (B) Cells were infected at an MOI of 1 for 10 min with doubly labeled rcTLPs (VP7, Atto 565; DLP, Atto 647N) and washed, and infection was allowed to continue for 6 h. Paraformaldehyde fixation was followed by incubation with a primary antibody that recognizes NSP2, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then imaged at 640-nm (left), 561-nm (middle), and 488-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (561-nm channel), colocalized with a strong signal in the 488-nm channel, corresponding to the presence of NSP2. Green boxes, similarly large NSP2 clusters that do not colocalize with DLP signal.
    Figure Legend Snippet: Viroplasm localization with respect to fluorescently labeled DLPs. (A) Cells were infected at an MOI of 5 for 10 min with doubly labeled rcTLPs (VP7, Atto 488; DLP, Atto 647N) and washed, and infection was allowed to continue for 5 h. Paraformaldehyde fixation was followed by overnight incubation with the pool of 44 Atto 565-labeled oligonucleotide probes and subsequent 3D imaging at 640-nm (left), 488-nm (middle), and 561-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (488-nm channel), colocalized with a strong signal in the Atto 565 oligonucleotide channel (561 nm). Green boxes, similarly large oligonucleotide bodies that do not colocalize with any DLP signal. (B) Cells were infected at an MOI of 1 for 10 min with doubly labeled rcTLPs (VP7, Atto 565; DLP, Atto 647N) and washed, and infection was allowed to continue for 6 h. Paraformaldehyde fixation was followed by incubation with a primary antibody that recognizes NSP2, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then imaged at 640-nm (left), 561-nm (middle), and 488-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (561-nm channel), colocalized with a strong signal in the 488-nm channel, corresponding to the presence of NSP2. Green boxes, similarly large NSP2 clusters that do not colocalize with DLP signal.

    Techniques Used: Labeling, Infection, Incubation, Imaging

    In vitro mRNA synthesis and labeled oligonucleotide hybridization. (A) Unlabeled DLPs were incubated with (top) or without (bottom) nucleotides to allow for mRNA production, followed by incubation with a primary VP6-specific antibody and a secondary Alexa 488-conjugated goat anti-mouse IgG. The samples were then incubated with the Atto 565 oligonucleotide probe pool and subsequently imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red) and 561-nm (green) channels are shown. White arrows, DLP/oligonucleotide colocalized particles (pseudocolored yellow); green arrows, DLP signal alone (pseudocolored red). (B) Scatter plot of the number of Atto 565 oligonucleotides colocalized with Alexa 488 signal from samples with (○) and without (□) nucleotides. From samples incubated with the required nucleotides, 7,765 Alexa 488-containing particles colocalized with 4 or more Atto 565 oligonucleotide probes, with an average of ∼20 probes per particle. Conversely, only 45 particles in samples incubated without nucleotides had any significant colocalized oligonucleotide signal. (C) Atto 647N-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with the Atto 565 oligonucleotide probe pool, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 640-nm (red) and 561-nm (green) channels are shown. (D) Probability density of 640-nm channel amplitudes. Detections with amplitudes less than 1,500 were not considered DLPs; detections above 4,000 were considered aggregates or multiple, unresolvable DLPs. (E) Scatter plot of the number of Atto 565 oligonucleotides colocalized with DLPs from samples with and without nucleotides. Of the 1,332 DLPs incubated with nucleotides, 1,291 (96.9%) colocalized with an average of ∼21 Atto 565 oligonucleotide probes (○), while 41 (3.1%) had no significant colocalization of probe. Of the 607 DLPs incubated without nucleotides, 602 (99.2%) had no significant colocalization of probe and 5 (0.8%) did colocalize (□). (F) Atto 488-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with both the Atto 565 and Atto 647N oligonucleotide probe pools, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red), 561-nm (green), and 640-nm (blue) channels are shown. (G) Probability density of 488-nm channel amplitudes derived from image analysis. Detections with amplitudes of less than 400 were not considered DLPs; detections above 1,500 were considered aggregates or multiple unresolvable DLPs. (H) Scatter plot of the number of Atto 565 and Atto 647N oligonucleotides colocalized with DLPs from the samples for panel D. Of a total of 1,126 analyzed DLPs incubated with nucleotides, 1,038 (92.2%) colocalized with both probes (○), 53 (4.7%) colocalized with only the Atto 565 probe (□, black), 14 (1.2%) colocalized with only the Atto 647N probe (△, black), and 21 (1.9%) had no significant colocalization of probe. Of 152 DLPs incubated without nucleotides analyzed, 144 (94.7%) had no significant colocalization of probe, 6 (3.9%) colocalized with only the Atto 565 probe (□, red), and 2 (1.3%), colocalized with only the Atto 647N probe (△, red). Amplitude detection and quantification of the number of labeled probes were performed as described in Materials and Methods.
    Figure Legend Snippet: In vitro mRNA synthesis and labeled oligonucleotide hybridization. (A) Unlabeled DLPs were incubated with (top) or without (bottom) nucleotides to allow for mRNA production, followed by incubation with a primary VP6-specific antibody and a secondary Alexa 488-conjugated goat anti-mouse IgG. The samples were then incubated with the Atto 565 oligonucleotide probe pool and subsequently imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red) and 561-nm (green) channels are shown. White arrows, DLP/oligonucleotide colocalized particles (pseudocolored yellow); green arrows, DLP signal alone (pseudocolored red). (B) Scatter plot of the number of Atto 565 oligonucleotides colocalized with Alexa 488 signal from samples with (○) and without (□) nucleotides. From samples incubated with the required nucleotides, 7,765 Alexa 488-containing particles colocalized with 4 or more Atto 565 oligonucleotide probes, with an average of ∼20 probes per particle. Conversely, only 45 particles in samples incubated without nucleotides had any significant colocalized oligonucleotide signal. (C) Atto 647N-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with the Atto 565 oligonucleotide probe pool, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 640-nm (red) and 561-nm (green) channels are shown. (D) Probability density of 640-nm channel amplitudes. Detections with amplitudes less than 1,500 were not considered DLPs; detections above 4,000 were considered aggregates or multiple, unresolvable DLPs. (E) Scatter plot of the number of Atto 565 oligonucleotides colocalized with DLPs from samples with and without nucleotides. Of the 1,332 DLPs incubated with nucleotides, 1,291 (96.9%) colocalized with an average of ∼21 Atto 565 oligonucleotide probes (○), while 41 (3.1%) had no significant colocalization of probe. Of the 607 DLPs incubated without nucleotides, 602 (99.2%) had no significant colocalization of probe and 5 (0.8%) did colocalize (□). (F) Atto 488-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with both the Atto 565 and Atto 647N oligonucleotide probe pools, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red), 561-nm (green), and 640-nm (blue) channels are shown. (G) Probability density of 488-nm channel amplitudes derived from image analysis. Detections with amplitudes of less than 400 were not considered DLPs; detections above 1,500 were considered aggregates or multiple unresolvable DLPs. (H) Scatter plot of the number of Atto 565 and Atto 647N oligonucleotides colocalized with DLPs from the samples for panel D. Of a total of 1,126 analyzed DLPs incubated with nucleotides, 1,038 (92.2%) colocalized with both probes (○), 53 (4.7%) colocalized with only the Atto 565 probe (□, black), 14 (1.2%) colocalized with only the Atto 647N probe (△, black), and 21 (1.9%) had no significant colocalization of probe. Of 152 DLPs incubated without nucleotides analyzed, 144 (94.7%) had no significant colocalization of probe, 6 (3.9%) colocalized with only the Atto 565 probe (□, red), and 2 (1.3%), colocalized with only the Atto 647N probe (△, red). Amplitude detection and quantification of the number of labeled probes were performed as described in Materials and Methods.

    Techniques Used: In Vitro, Labeling, Hybridization, Incubation, Derivative Assay

    31) Product Images from "Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis"

    Article Title: Identification of a pathogenic antibody response to native myelin oligodendrocyte glycoprotein in multiple sclerosis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0607242103

    Human antibodies to native MOG bind to intact myelin. Rat brain slices were stained with 8–18C5 mAb ( a and d ), anti-MOG antibody-positive and -negative sera. Staining was visualized by an anti-IgG antibody labeled with Alexa Fluor 488. Stainings of one representative anti-MOG antibody-positive serum of five ( b and e ) and one of four negative sera ( c ) are shown. (Magnification: a – c , ×100; d and e , ×200.)
    Figure Legend Snippet: Human antibodies to native MOG bind to intact myelin. Rat brain slices were stained with 8–18C5 mAb ( a and d ), anti-MOG antibody-positive and -negative sera. Staining was visualized by an anti-IgG antibody labeled with Alexa Fluor 488. Stainings of one representative anti-MOG antibody-positive serum of five ( b and e ) and one of four negative sera ( c ) are shown. (Magnification: a – c , ×100; d and e , ×200.)

    Techniques Used: Staining, Labeling

    32) Product Images from "Absence of Spontaneous Central Nervous System Remyelination in Class II-deficient Mice Infected with Theiler’s Virus"

    Article Title: Absence of Spontaneous Central Nervous System Remyelination in Class II-deficient Mice Infected with Theiler’s Virus

    Journal: Journal of neuropathology and experimental neurology

    doi:

    Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β 2 m [−/−]) mice. Total and TMEV-specific IgG and IgM in serum of chronically TMEV-infected Ab° (N = 6), β 2 m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β 2 m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β 2 m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β 2 m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β 2 m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.
    Figure Legend Snippet: Total and virus-specific antibodies in class II-deficient (Ab°) and class I-deficient β 2 m [−/−]) mice. Total and TMEV-specific IgG and IgM in serum of chronically TMEV-infected Ab° (N = 6), β 2 m (−/−) (N = 4), and nonmutant C57BL/6 (N = 3) mice were determined by indirect ELISA. (A) Total IgG in Ab° was comparable to nonmutant C57BL/6 whereas (B) IgM antibodies were higher in Ab° mice than in C57BL/6 and β 2 m (−/−) mice. (C) TMEV-specific IgG was not present in Ab° mice but was detected in C57BL/6 and β 2 m (−/−) mice. (D) Ab° mice showed TMEV-specific IgM which was not observed in nonmutant C57BL/6 and β 2 m (−/−) mice. Because sera from Ab° mice did not neutralize TMEV in vitro (Insert in D), whereas sera from C57BL/6 and β 2 m (−/−) did, this indicated that the TMEV-neutralizing antibodies are predominantly IgG. Pooled serum from noninfected C57BL/6 mice was used as a negative control.

    Techniques Used: Mouse Assay, Infection, Indirect ELISA, In Vitro, Negative Control

    33) Product Images from "Protein Tyrosine Kinase Signaling in the Mouse Oocyte Cortex During Sperm-Egg Interactions and Anaphase Resumption"

    Article Title: Protein Tyrosine Kinase Signaling in the Mouse Oocyte Cortex During Sperm-Egg Interactions and Anaphase Resumption

    Journal: Molecular reproduction and development

    doi: 10.1002/mrd.22160

    Detection and sub-cellular distribution of PYK2 and FAK kinases Western blot analysis of MII oocytes (left) was performed on groups of 60 oocytes/ lane probed with antibodies to FAK protein (FAK protein), antibodies to phosphorylated FAK (FAK PY 861 ), anti PYK2 protein (PYK2 protein), or anti phosphorylated PYK2 (PYK2 PY 579 ) as described in’ Materials and Methods’. The apparent molecular weight of each major band was calculated by comparison with molecular weight standards and is presented in the margins (arrows). Sub-cellular localization of FAK and PYK2 protein (right) was performed on oocytes collected prior to fertilization in vitro (A, C) or during anaphase/ telophase II (B, D) then labeled with anti-PYK2 protein and anti-FAK protein antibodies not targeted to phosphorylation sites in order to establish the sub-cellular distribution of the entire pool of these kinases in the oocyte. Bound antibodies were detected with alexa 488-anti-rabbit IgG (green). Chromatin was stained with ethidium homodimer (red) or Hoechst 33258 (blue). Magnification is indicated by the bar which represents 10 μm.
    Figure Legend Snippet: Detection and sub-cellular distribution of PYK2 and FAK kinases Western blot analysis of MII oocytes (left) was performed on groups of 60 oocytes/ lane probed with antibodies to FAK protein (FAK protein), antibodies to phosphorylated FAK (FAK PY 861 ), anti PYK2 protein (PYK2 protein), or anti phosphorylated PYK2 (PYK2 PY 579 ) as described in’ Materials and Methods’. The apparent molecular weight of each major band was calculated by comparison with molecular weight standards and is presented in the margins (arrows). Sub-cellular localization of FAK and PYK2 protein (right) was performed on oocytes collected prior to fertilization in vitro (A, C) or during anaphase/ telophase II (B, D) then labeled with anti-PYK2 protein and anti-FAK protein antibodies not targeted to phosphorylation sites in order to establish the sub-cellular distribution of the entire pool of these kinases in the oocyte. Bound antibodies were detected with alexa 488-anti-rabbit IgG (green). Chromatin was stained with ethidium homodimer (red) or Hoechst 33258 (blue). Magnification is indicated by the bar which represents 10 μm.

    Techniques Used: Western Blot, Molecular Weight, In Vitro, Labeling, Staining

    34) Product Images from "Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)"

    Article Title: Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

    Journal: bioRxiv

    doi: 10.1101/2020.03.12.988923

    Re-embedding enables Ex- d STORM. a , Model of microtubules with an outer diameter of 25 nm stained with conventional primary (pab) and fluorescently labeled secondary IgG antibodies (sab) results in a total diameter of 60 nm with a linkage error (defined by the size of the primary and secondary antibody) of 17.5 nm 22 . b , d STORM image of pre-labeled proExM expanded and re-embedded Cos-7 cells stained with primary antibodies against α-tubulin and secondary Alexa Fluor 532 conjugated antibodies (Al532). The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion (pre-labeled). c , Zoom in on highlighted region in (b). d , Averaged cross-sectional profile of 9 microtubule segments with a total length of 29.1 µm (segment lengths range from 2.1-4.5 µm) measured in 2 cells from 1 expanded sample. e , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of the data analyzed in (c) with an average distance of 137.1 ± 10.1 nm (mean ± sd). f , Unexpanded Cos-7 cells labeled with an anti α-tubulin primary antibody and Alexa Fluor 532 (Al532) conjugated IgG secondary antibodies. The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded. g , Zoom in of the white boxed region in (f). h , Average intensity profile of 35 microtubule segments with a length between 1.1 – 5.8 µm (mean = 2.0 µm) and a total length of 69.6 µm analyzed in 12 d STORM images. d , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of cross-sectional profiles of the analyzed microtubule segments in (h) with a mean peak-to-peak distance of 36.2 ± 5.4 nm (mean ± sd). Scale bars, 2 µm (b,f), 500 nm (c,g).
    Figure Legend Snippet: Re-embedding enables Ex- d STORM. a , Model of microtubules with an outer diameter of 25 nm stained with conventional primary (pab) and fluorescently labeled secondary IgG antibodies (sab) results in a total diameter of 60 nm with a linkage error (defined by the size of the primary and secondary antibody) of 17.5 nm 22 . b , d STORM image of pre-labeled proExM expanded and re-embedded Cos-7 cells stained with primary antibodies against α-tubulin and secondary Alexa Fluor 532 conjugated antibodies (Al532). The small logo in the upper left corner symbolizes that microtubules have been immunolabeled before expansion (pre-labeled). c , Zoom in on highlighted region in (b). d , Averaged cross-sectional profile of 9 microtubule segments with a total length of 29.1 µm (segment lengths range from 2.1-4.5 µm) measured in 2 cells from 1 expanded sample. e , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of the data analyzed in (c) with an average distance of 137.1 ± 10.1 nm (mean ± sd). f , Unexpanded Cos-7 cells labeled with an anti α-tubulin primary antibody and Alexa Fluor 532 (Al532) conjugated IgG secondary antibodies. The small logo in the upper left corner symbolizes that microtubules have been immunolabeled and not expanded. g , Zoom in of the white boxed region in (f). h , Average intensity profile of 35 microtubule segments with a length between 1.1 – 5.8 µm (mean = 2.0 µm) and a total length of 69.6 µm analyzed in 12 d STORM images. d , Histogram of peak-to-peak distances determined by bi-Gaussian fitting of cross-sectional profiles of the analyzed microtubule segments in (h) with a mean peak-to-peak distance of 36.2 ± 5.4 nm (mean ± sd). Scale bars, 2 µm (b,f), 500 nm (c,g).

    Techniques Used: Staining, Labeling, Immunolabeling

    Pre-labeling Ex- d STORM. a , Simulated intensity profiles using a cylindrical distribution function to describe unexpanded or 3.2x expanded immunostained microtubules (labeled with IgG antibodies or DNA modified IgG antibodies pre-expansion) and resulting peak-to-peak distances of the cross-sectional profiles. b , d STORM image of expanded and re-embedded α− and β-tubulin pre-labeled with secondary Alexa Fluor 532 IgG antibodies (Al532) using the MA-NHS/GA method 6 , i.e. antibodies are cross-linked with glutaraldehyde (GA) into the hydrogel (Antibody-Al532 (GA)). c , Zoom in of white boxed region in (b). d , Averaged cross-sectional profile of 8 microtubule segments with a length between 1.5 - 6.4 µm and 28.6 µm in total measured in 4 expanded cells. e , Histogram of peak-to-peak distance distribution of microtubule segments analyzed in (d) with a mean distance of 133.8 ± 13.2 nm (mean ± sd). f , Unexpanded d STORM image of ssDNA-Cy5 secondary antibody hybridized with Cy5 bearing oligonucleotides pre-expansion (DNA-Cy5 protocol). g , Magnified view of white boxed region in (f). h , Average cross-sectional profile of 7 microtubule segments with a length between 1.0 – 1.8 µm and 8.7 µm in total. i , Histogram of peak-to-peak distances of the data in (h) with a mean distance of 43.9 ± 3.7 nm (mean ± sd). j , Expanded d STORM image of microtubules labeled with α-tubulin and dsDNA (DNA-Al532) conjugated secondary antibodies exhibiting a methacryloyl group to crosslink the DNA with fluorophores pre-expansion into the hydrogel (original ExM trifunctional label concept) 1 . k , Zoom-in of white boxed region in (j). l , Average intensity profile of 26 microtubule segments with a length of 2.4 – 10.7 µm and 118.6 µm in total. m , Histogram of peak-to-peak distances determined from microtubule segments in (l) with a mean distance of 226.7 ± 15.3 nm (mean ± sd). n , d STORM image α− and β-tubulin expanded according the DNA-Cy5 protocol strategy with labels at Cy5-bearing oligonucleotides introduced post-re-embedding. o , Zoom in of white boxed region in (n). p , Average intensity profile of 15 microtubule segments with a length between 1.6-25.1 µm and a total length of 126.0 µm in 1 expanded sample. q , Histogram of peak-to-peak distances determined by fitting the cross-sectional profiles analyzed in (p) with a mean distance of 201.0 ± 9.3 nm (mean ± sd). The small logos in the upper left corner symbolize the labeling method, e.g. pre- and post-immunolabeled with or without DNA-linker, respectively. Scale bars, 2 µm (b,f,j,n), 500 nm (c,g,k,o).
    Figure Legend Snippet: Pre-labeling Ex- d STORM. a , Simulated intensity profiles using a cylindrical distribution function to describe unexpanded or 3.2x expanded immunostained microtubules (labeled with IgG antibodies or DNA modified IgG antibodies pre-expansion) and resulting peak-to-peak distances of the cross-sectional profiles. b , d STORM image of expanded and re-embedded α− and β-tubulin pre-labeled with secondary Alexa Fluor 532 IgG antibodies (Al532) using the MA-NHS/GA method 6 , i.e. antibodies are cross-linked with glutaraldehyde (GA) into the hydrogel (Antibody-Al532 (GA)). c , Zoom in of white boxed region in (b). d , Averaged cross-sectional profile of 8 microtubule segments with a length between 1.5 - 6.4 µm and 28.6 µm in total measured in 4 expanded cells. e , Histogram of peak-to-peak distance distribution of microtubule segments analyzed in (d) with a mean distance of 133.8 ± 13.2 nm (mean ± sd). f , Unexpanded d STORM image of ssDNA-Cy5 secondary antibody hybridized with Cy5 bearing oligonucleotides pre-expansion (DNA-Cy5 protocol). g , Magnified view of white boxed region in (f). h , Average cross-sectional profile of 7 microtubule segments with a length between 1.0 – 1.8 µm and 8.7 µm in total. i , Histogram of peak-to-peak distances of the data in (h) with a mean distance of 43.9 ± 3.7 nm (mean ± sd). j , Expanded d STORM image of microtubules labeled with α-tubulin and dsDNA (DNA-Al532) conjugated secondary antibodies exhibiting a methacryloyl group to crosslink the DNA with fluorophores pre-expansion into the hydrogel (original ExM trifunctional label concept) 1 . k , Zoom-in of white boxed region in (j). l , Average intensity profile of 26 microtubule segments with a length of 2.4 – 10.7 µm and 118.6 µm in total. m , Histogram of peak-to-peak distances determined from microtubule segments in (l) with a mean distance of 226.7 ± 15.3 nm (mean ± sd). n , d STORM image α− and β-tubulin expanded according the DNA-Cy5 protocol strategy with labels at Cy5-bearing oligonucleotides introduced post-re-embedding. o , Zoom in of white boxed region in (n). p , Average intensity profile of 15 microtubule segments with a length between 1.6-25.1 µm and a total length of 126.0 µm in 1 expanded sample. q , Histogram of peak-to-peak distances determined by fitting the cross-sectional profiles analyzed in (p) with a mean distance of 201.0 ± 9.3 nm (mean ± sd). The small logos in the upper left corner symbolize the labeling method, e.g. pre- and post-immunolabeled with or without DNA-linker, respectively. Scale bars, 2 µm (b,f,j,n), 500 nm (c,g,k,o).

    Techniques Used: Labeling, Modification, Immunolabeling

    35) Product Images from "Single-Particle Detection of Transcription following Rotavirus Entry"

    Article Title: Single-Particle Detection of Transcription following Rotavirus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.00651-17

    NSP2 and Atto 565 oligonucleotide probe colocalization in TLP-infected cells. BSC-1 cells were infected at an MOI of 20 (top row) or mock infected (bottom row). Infection was allowed to proceed at 37°C for 6 h. After paraformaldehyde fixation, the cells were permeabilized with 1% Triton X-100 and probed with a primary, NSP2-specific antibody, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then incubated overnight with the pool of Atto 565-labeled oligonucleotides and imaged. Maximum-intensity z-projections of the 488-nm channel (left), the 561-nm channel (middle), and the overlay of the two channels (right) are shown.
    Figure Legend Snippet: NSP2 and Atto 565 oligonucleotide probe colocalization in TLP-infected cells. BSC-1 cells were infected at an MOI of 20 (top row) or mock infected (bottom row). Infection was allowed to proceed at 37°C for 6 h. After paraformaldehyde fixation, the cells were permeabilized with 1% Triton X-100 and probed with a primary, NSP2-specific antibody, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then incubated overnight with the pool of Atto 565-labeled oligonucleotides and imaged. Maximum-intensity z-projections of the 488-nm channel (left), the 561-nm channel (middle), and the overlay of the two channels (right) are shown.

    Techniques Used: Infection, Incubation, Labeling

    Viroplasm localization with respect to fluorescently labeled DLPs. (A) Cells were infected at an MOI of 5 for 10 min with doubly labeled rcTLPs (VP7, Atto 488; DLP, Atto 647N) and washed, and infection was allowed to continue for 5 h. Paraformaldehyde fixation was followed by overnight incubation with the pool of 44 Atto 565-labeled oligonucleotide probes and subsequent 3D imaging at 640-nm (left), 488-nm (middle), and 561-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (488-nm channel), colocalized with a strong signal in the Atto 565 oligonucleotide channel (561 nm). Green boxes, similarly large oligonucleotide bodies that do not colocalize with any DLP signal. (B) Cells were infected at an MOI of 1 for 10 min with doubly labeled rcTLPs (VP7, Atto 565; DLP, Atto 647N) and washed, and infection was allowed to continue for 6 h. Paraformaldehyde fixation was followed by incubation with a primary antibody that recognizes NSP2, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then imaged at 640-nm (left), 561-nm (middle), and 488-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (561-nm channel), colocalized with a strong signal in the 488-nm channel, corresponding to the presence of NSP2. Green boxes, similarly large NSP2 clusters that do not colocalize with DLP signal.
    Figure Legend Snippet: Viroplasm localization with respect to fluorescently labeled DLPs. (A) Cells were infected at an MOI of 5 for 10 min with doubly labeled rcTLPs (VP7, Atto 488; DLP, Atto 647N) and washed, and infection was allowed to continue for 5 h. Paraformaldehyde fixation was followed by overnight incubation with the pool of 44 Atto 565-labeled oligonucleotide probes and subsequent 3D imaging at 640-nm (left), 488-nm (middle), and 561-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (488-nm channel), colocalized with a strong signal in the Atto 565 oligonucleotide channel (561 nm). Green boxes, similarly large oligonucleotide bodies that do not colocalize with any DLP signal. (B) Cells were infected at an MOI of 1 for 10 min with doubly labeled rcTLPs (VP7, Atto 565; DLP, Atto 647N) and washed, and infection was allowed to continue for 6 h. Paraformaldehyde fixation was followed by incubation with a primary antibody that recognizes NSP2, followed by incubation with a secondary IgG coupled to Alexa 488. The samples were then imaged at 640-nm (left), 561-nm (middle), and 488-nm (right) excitation wavelengths. Maximum-intensity projections of all three channels are shown. Red boxes, uncoated DLPs (640-nm channel), as indicated by the lack of VP7 (561-nm channel), colocalized with a strong signal in the 488-nm channel, corresponding to the presence of NSP2. Green boxes, similarly large NSP2 clusters that do not colocalize with DLP signal.

    Techniques Used: Labeling, Infection, Incubation, Imaging

    In vitro mRNA synthesis and labeled oligonucleotide hybridization. (A) Unlabeled DLPs were incubated with (top) or without (bottom) nucleotides to allow for mRNA production, followed by incubation with a primary VP6-specific antibody and a secondary Alexa 488-conjugated goat anti-mouse IgG. The samples were then incubated with the Atto 565 oligonucleotide probe pool and subsequently imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red) and 561-nm (green) channels are shown. White arrows, DLP/oligonucleotide colocalized particles (pseudocolored yellow); green arrows, DLP signal alone (pseudocolored red). (B) Scatter plot of the number of Atto 565 oligonucleotides colocalized with Alexa 488 signal from samples with (○) and without (□) nucleotides. From samples incubated with the required nucleotides, 7,765 Alexa 488-containing particles colocalized with 4 or more Atto 565 oligonucleotide probes, with an average of ∼20 probes per particle. Conversely, only 45 particles in samples incubated without nucleotides had any significant colocalized oligonucleotide signal. (C) Atto 647N-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with the Atto 565 oligonucleotide probe pool, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 640-nm (red) and 561-nm (green) channels are shown. (D) Probability density of 640-nm channel amplitudes. Detections with amplitudes less than 1,500 were not considered DLPs; detections above 4,000 were considered aggregates or multiple, unresolvable DLPs. (E) Scatter plot of the number of Atto 565 oligonucleotides colocalized with DLPs from samples with and without nucleotides. Of the 1,332 DLPs incubated with nucleotides, 1,291 (96.9%) colocalized with an average of ∼21 Atto 565 oligonucleotide probes (○), while 41 (3.1%) had no significant colocalization of probe. Of the 607 DLPs incubated without nucleotides, 602 (99.2%) had no significant colocalization of probe and 5 (0.8%) did colocalize (□). (F) Atto 488-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with both the Atto 565 and Atto 647N oligonucleotide probe pools, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red), 561-nm (green), and 640-nm (blue) channels are shown. (G) Probability density of 488-nm channel amplitudes derived from image analysis. Detections with amplitudes of less than 400 were not considered DLPs; detections above 1,500 were considered aggregates or multiple unresolvable DLPs. (H) Scatter plot of the number of Atto 565 and Atto 647N oligonucleotides colocalized with DLPs from the samples for panel D. Of a total of 1,126 analyzed DLPs incubated with nucleotides, 1,038 (92.2%) colocalized with both probes (○), 53 (4.7%) colocalized with only the Atto 565 probe (□, black), 14 (1.2%) colocalized with only the Atto 647N probe (△, black), and 21 (1.9%) had no significant colocalization of probe. Of 152 DLPs incubated without nucleotides analyzed, 144 (94.7%) had no significant colocalization of probe, 6 (3.9%) colocalized with only the Atto 565 probe (□, red), and 2 (1.3%), colocalized with only the Atto 647N probe (△, red). Amplitude detection and quantification of the number of labeled probes were performed as described in Materials and Methods.
    Figure Legend Snippet: In vitro mRNA synthesis and labeled oligonucleotide hybridization. (A) Unlabeled DLPs were incubated with (top) or without (bottom) nucleotides to allow for mRNA production, followed by incubation with a primary VP6-specific antibody and a secondary Alexa 488-conjugated goat anti-mouse IgG. The samples were then incubated with the Atto 565 oligonucleotide probe pool and subsequently imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red) and 561-nm (green) channels are shown. White arrows, DLP/oligonucleotide colocalized particles (pseudocolored yellow); green arrows, DLP signal alone (pseudocolored red). (B) Scatter plot of the number of Atto 565 oligonucleotides colocalized with Alexa 488 signal from samples with (○) and without (□) nucleotides. From samples incubated with the required nucleotides, 7,765 Alexa 488-containing particles colocalized with 4 or more Atto 565 oligonucleotide probes, with an average of ∼20 probes per particle. Conversely, only 45 particles in samples incubated without nucleotides had any significant colocalized oligonucleotide signal. (C) Atto 647N-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with the Atto 565 oligonucleotide probe pool, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 640-nm (red) and 561-nm (green) channels are shown. (D) Probability density of 640-nm channel amplitudes. Detections with amplitudes less than 1,500 were not considered DLPs; detections above 4,000 were considered aggregates or multiple, unresolvable DLPs. (E) Scatter plot of the number of Atto 565 oligonucleotides colocalized with DLPs from samples with and without nucleotides. Of the 1,332 DLPs incubated with nucleotides, 1,291 (96.9%) colocalized with an average of ∼21 Atto 565 oligonucleotide probes (○), while 41 (3.1%) had no significant colocalization of probe. Of the 607 DLPs incubated without nucleotides, 602 (99.2%) had no significant colocalization of probe and 5 (0.8%) did colocalize (□). (F) Atto 488-labeled DLPs were incubated with (top) or without (bottom) nucleotides. Following incubation with both the Atto 565 and Atto 647N oligonucleotide probe pools, DLPs were imaged in 3D. Maximum-intensity z-projections of the overlay of the 488-nm (red), 561-nm (green), and 640-nm (blue) channels are shown. (G) Probability density of 488-nm channel amplitudes derived from image analysis. Detections with amplitudes of less than 400 were not considered DLPs; detections above 1,500 were considered aggregates or multiple unresolvable DLPs. (H) Scatter plot of the number of Atto 565 and Atto 647N oligonucleotides colocalized with DLPs from the samples for panel D. Of a total of 1,126 analyzed DLPs incubated with nucleotides, 1,038 (92.2%) colocalized with both probes (○), 53 (4.7%) colocalized with only the Atto 565 probe (□, black), 14 (1.2%) colocalized with only the Atto 647N probe (△, black), and 21 (1.9%) had no significant colocalization of probe. Of 152 DLPs incubated without nucleotides analyzed, 144 (94.7%) had no significant colocalization of probe, 6 (3.9%) colocalized with only the Atto 565 probe (□, red), and 2 (1.3%), colocalized with only the Atto 647N probe (△, red). Amplitude detection and quantification of the number of labeled probes were performed as described in Materials and Methods.

    Techniques Used: In Vitro, Labeling, Hybridization, Incubation, Derivative Assay

    36) Product Images from "Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils"

    Article Title: Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils

    Journal: Mediators of Inflammation

    doi: 10.1155/2018/4847205

    Flow cytometric analysis of antiapoptotic effects of SP and CGRP towards A549 cell death induced by M. catarrhalis OMVs. (a) The A549 cells were treated with various stimulants for 24 h of incubation, processed, stained with Annexin V and PI antibodies, and analyzed using Muse Cell Analyzer (Merck). The upper panel refers to untreated control cells and positive control (5 mM H 2 O 2 ). The lower panel refers to the cells treated with OMVs 100 μ g/ml alone or in the presence of 10 −8 M of SP or CGRP. Representative data from at least two independent experiments are shown. (b) Cell surface expression of CEACAM1 in A549 obtained from 1-day postconfluent phase of growth. A549 cells were stained for CEACAM1 with mAb clone 283340 as primary antibody or isotype-matched control followed by Alexa Fluor 488-conjugated goat anti-mouse IgG superclonal secondary antibody. Subsequently, samples were measured by FACs. The data shown are representative of two independent but essentially the same experiments performed in duplicates.
    Figure Legend Snippet: Flow cytometric analysis of antiapoptotic effects of SP and CGRP towards A549 cell death induced by M. catarrhalis OMVs. (a) The A549 cells were treated with various stimulants for 24 h of incubation, processed, stained with Annexin V and PI antibodies, and analyzed using Muse Cell Analyzer (Merck). The upper panel refers to untreated control cells and positive control (5 mM H 2 O 2 ). The lower panel refers to the cells treated with OMVs 100 μ g/ml alone or in the presence of 10 −8 M of SP or CGRP. Representative data from at least two independent experiments are shown. (b) Cell surface expression of CEACAM1 in A549 obtained from 1-day postconfluent phase of growth. A549 cells were stained for CEACAM1 with mAb clone 283340 as primary antibody or isotype-matched control followed by Alexa Fluor 488-conjugated goat anti-mouse IgG superclonal secondary antibody. Subsequently, samples were measured by FACs. The data shown are representative of two independent but essentially the same experiments performed in duplicates.

    Techniques Used: Flow Cytometry, Incubation, Staining, Positive Control, Expressing, FACS

    37) Product Images from "Aquaporin 11, a regulator of water efflux at retinal Müller glial cell surface decreases concomitant with immune-mediated gliosis"

    Article Title: Aquaporin 11, a regulator of water efflux at retinal Müller glial cell surface decreases concomitant with immune-mediated gliosis

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0554-2

    Assessment of AQP11 expression with immunohistochemistry, scale bars = 20 μm. a Expression in equine retina. AQP11 ( red ) was specifically expressed at retinal Müller glial cells, reaching from endfeet in ganglion cell layer to outer limiting membrane; ILM inner limiting membrane ( arrow ), GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, OLM outer limiting membrane ( arrowhead ). b Negative control, incubated only with secondary antibody anti mouse IgG Alexa 568. c Overlay of Müller glia marker vimentin ( green ) and AQP11 expression ( red ) in equine retina results in yellow color. d Müller glia cell membrane localization of AQP11 ( violet ) stained with fine-grained resolution. e Preincubation of AQP11 antibody with 100 μg/ml irrelevant peptide did not interfere with antibody binding, AQP11: red . f Preincubation with 100 μg/ml AQP11 immunization peptide completely blocked antibody binding. g Preincubation with 1 μg/ml AQP11 immunization peptide partly blocked antibody binding. h Müller glia endfeet of mouse and i rat retina express AQP11 ( red ). j AQP11 ( red ) expression in positive control tissue mouse kidney
    Figure Legend Snippet: Assessment of AQP11 expression with immunohistochemistry, scale bars = 20 μm. a Expression in equine retina. AQP11 ( red ) was specifically expressed at retinal Müller glial cells, reaching from endfeet in ganglion cell layer to outer limiting membrane; ILM inner limiting membrane ( arrow ), GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, OLM outer limiting membrane ( arrowhead ). b Negative control, incubated only with secondary antibody anti mouse IgG Alexa 568. c Overlay of Müller glia marker vimentin ( green ) and AQP11 expression ( red ) in equine retina results in yellow color. d Müller glia cell membrane localization of AQP11 ( violet ) stained with fine-grained resolution. e Preincubation of AQP11 antibody with 100 μg/ml irrelevant peptide did not interfere with antibody binding, AQP11: red . f Preincubation with 100 μg/ml AQP11 immunization peptide completely blocked antibody binding. g Preincubation with 1 μg/ml AQP11 immunization peptide partly blocked antibody binding. h Müller glia endfeet of mouse and i rat retina express AQP11 ( red ). j AQP11 ( red ) expression in positive control tissue mouse kidney

    Techniques Used: Expressing, Immunohistochemistry, Negative Control, Incubation, Marker, Staining, Binding Assay, Positive Control

    38) Product Images from "Vaccinia Virus F9 Virion Membrane Protein Is Required for Entry but Not Virus Assembly, in Contrast to the Related L1 Protein"

    Article Title: Vaccinia Virus F9 Virion Membrane Protein Is Required for Entry but Not Virus Assembly, in Contrast to the Related L1 Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01149-06

    Cell binding and penetration assay. HeLa cells were incubated with 10 PFU of purified F9 + virions or the same number of F9 − virions at 4°C for 1 h and then shifted to 37°C for 2 h. Subsequently, cells were fixed, permeabilized, and stained for indirect immunofluorescence microscopy. The L1 MV membrane protein was stained with a mouse monoclonal anti-L1 antibody and a goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (green). The A4 core protein was stained with a rabbit primary antibody and a goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 568 (red). Cellular DNA was stained with DAPI (blue).
    Figure Legend Snippet: Cell binding and penetration assay. HeLa cells were incubated with 10 PFU of purified F9 + virions or the same number of F9 − virions at 4°C for 1 h and then shifted to 37°C for 2 h. Subsequently, cells were fixed, permeabilized, and stained for indirect immunofluorescence microscopy. The L1 MV membrane protein was stained with a mouse monoclonal anti-L1 antibody and a goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (green). The A4 core protein was stained with a rabbit primary antibody and a goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 568 (red). Cellular DNA was stained with DAPI (blue).

    Techniques Used: Binding Assay, Incubation, Purification, Staining, Immunofluorescence, Microscopy

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    Article Snippet: .. Sections were stained for the presence of mouse IgG, rabbit IgG, and mouse C1q using goat anti-mouse IgG Oregon Green (Invitrogen Corp.), goat anti-rabbit IgG conjugated to FITC (Nordic Immunological Laboratories), and rabbit anti–mouse C1q DIG, followed by sheep anti-DIG conjugated to HRP, where appropriate. .. For double staining, goat anti-mouse IgG Alexa 546 (Invitrogen Corp.) was used in combination with anti-C1q and anti-rabbit IgG stainings, as described above.

    Article Title: Identification of the Mycobacterium ulcerans Protein MUL_3720 as a Promising Target for the Development of a Diagnostic Test for Buruli Ulcer
    Article Snippet: Agarose blocks were embedded into paraffin, cut in 3 μm sections and transferred onto microscopy glass slides (Thermo Scientific). .. Bacteria were stained with mAb JD3.2 and Alexa fluor488 (Invitrogen) conjugated goat anti-mouse IgG and mounted in ProLong Gold anti-fade reagent containing 4′,6-Diamidino-2-phenylindole (DAPI; Invitrogen). .. Mycobacterial protein fragment complementation The system described for investigating protein interactions by the functional reconstitution of a murine dehydrofolate reductase domain in M. tuberculosis [ ] was modified here for use in M. ulcerans.

    Incubation:

    Article Title: CaGdt1 plays a compensatory role for the calcium pump CaPmr1 in the regulation of calcium signaling and cell wall integrity signaling in Candida albicans
    Article Snippet: BSA of 400 μg ml− 1 was added to the spheroplast mixture, and incubated for 20 min before mouse anti-HA antibodies was added at a dilution of 1:500. .. The mixture was incubated for 2 h, and washed twice with PBST before goat anti-mouse IgG conjugated to Alexa Fluor 555 (Invitrogen, USA) was added. .. The mixture was incubated in dark for 45 min before spheroplasts were collected, washed three times, and visualized under a Nikon 80i microscope equipped with DS-U2 CCD.

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    Labeling:

    Article Title: RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors
    Article Snippet: Two primary antibodies were added simultaneously over the first night (one rabbit IgG, anti-Cav, and the other mouse IgG, anti-Cre) and then anti-CtBP2 was added for the second overnight incubation. .. In most instances, rabbit IgGs were labeled with chicken anti-rabbit IgG-Alexa Fluor 488 (Invitrogen, ) and mouse IgGs were labeled with goat anti-mouse IgG-Alexa Fluor 647 (Invitrogen, ). .. Confocal images of immunolabeled samples were collected as a stack of optical sections through the entire ∼10 μm cryosections and the images are presented here as maximal projections using ImageJ software.

    other:

    Article Title: A Soluble Fragment of the Tumor Antigen BCL2-associated Athanogene 6 (BAG-6) Is Essential and Sufficient for Inhibition of NKp30 Receptor-dependent Cytotoxicity of Natural Killer Cells *
    Article Snippet: The following antibodies were used: mouse monoclonal anti-polyhistidine tag HRP-conjugate and anti-human HRP-conjugate (both Sigma-Aldrich), anti-Strep-mAb classic HRP (IBA), anti-NKp30 clone p30-15 (kindly provided by Carsten Watzl), goat anti-mouse IgG1 APC conjugate (Life Technologies), and anti-CD4 APC conjugate (eBioscience).

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    • anti igm  (Thermo Fisher)
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    Thermo Fisher anti igm
    Phenotypic and functional characterization of the IL-33-induced Breg-like cells (Breg IL-33 ) in the blood. A–C. Surface phenotype of Breg IL-33 : The IL-33-induced IL-10 producing B cells expressed high surface <t>IgM</t> (A), CD1d and CD25 (B), but down-regulated CD23 (FceRII) (B, C). Data shown in the table under (B) were results showing the Mean MFI (±SD) values calculated from results of 5 repeated experiments, as ratio of the IL-10 − B, and IL-10 + B, cell subset over the total B cells gated. Statistical analysis: p values (Student t test). D–F. In vitro Breg suppression assays: Immunosuppressive effects of the IL-33-induced Breg-like cells on B effector (Beff) cell proliferation (D), division (E), and its IL-10 dependency (F). D. CD23 + B cells (Beff, 10 5 ) were cultured in the presence of <t>anti-CD40</t> (2.5 μg/ml), with titrating (as indicated) doses of CD23 − B cells (Breg IL-33 ) purified from the IL-33-injected mice. Cell proliferation was determined by thymidine incorporation at 48 h s. E. Breg IL-33 (CD23 - ) purified from hIL-33-injected WT mice (Wk-2) were primed with anti-CD40 (2.5 μg/ml) for 5 h before being added to CFSE-labelled CD23 + Beff cells. Cell division (CFSE dilution) was determined by flow cytometry at day 5 of culture in the presence or absence of LPS (0.5 μg/ml) or anti-IgM (5 μg/ml). F. IL-10 levels in the culture supernatants were quantified by ELISA (BD Biosciences).
    Anti Igm, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    alkaline phosphatase conjugated goat anti mouse igg  (Thermo Fisher)
    86
    Thermo Fisher alkaline phosphatase conjugated goat anti mouse igg
    Plasmodium falciparum EBA175RIII–V construct and expressed antigen. A schematic representation of the 1620 bp region of EBA175RIII–V cloned into the pLEA2 expression vector, which contains the nucleotide sequence of a hexahistidine tag inframe of the multiple cloning site ( a ). The culture supernatant (1) containing the secreted protein as well as the purified protein (2) was analysed by SDS-PAGE followed by coomassie staining ( b ) and a western blot probed with penta-His mouse monoclonal antibody <t>(IgG1)</t> ( c )
    Alkaline Phosphatase Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Confocal microscopy of cells labeled with anti–LDL receptor Ab. ( a – f ) EBV-lymphocytes were incubated with rabbit anti–LDL receptor (red) at 4°C, washed at 4°C, and either directly permeabilized ( a – c ) or incubated for 10 minutes at 37°C before permeabilization to allow internalization of LDL receptor/Ab complexes ( d – f ). Permeabilized cells were then incubated with mouse anti–α-adaptin (AP2), washed, and incubated with Alexa 568–conjugated goat anti-rabbit <t>IgG</t> (LDL receptors, red) and Alexa 488–conjugated goat anti-mouse IgG (AP2, green). Nuclei were stained with DAPI. The plates shown are an overlay of red and green images. The bar represents 5.0 μm. ( a and d ) Control cells; ( b and e ) cells from proband 1.1; ( c and f ) cells from proband 1.1 expressing viral c-myc-ARH. ( g – l ) EBV-lymphocytes ( g – i ) or cultured skin fibroblasts ( j – l ) from three different control subjects were incubated with anti–LDL receptor Ab at 4°C, permeabilized, and then incubated with anti–α-adaptin Ab (AP2) as described for a – f above. The bars represent 5.0 μm (in g for g – i , and in j for j – l ).
    Alexa 488 Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Role of DC-SIGN in the anti-DENV2 activity of bLF. The THP-1 cells were stimulated with recombinant IL-4, GM-CSF, and TNF-α according to a previous report [ 19 ], and dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expression was subsequently measured by flow cytometry using mouse anti-human DC-SIGN antibodies and fluorescein isothiocyanate <t>(FITC)-conjugated</t> goat anti-mouse immunoglobulin G <t>(IgG)</t> antibodies ( A ) The DC-SIGN-expressing and non-expessing THP-1 cells were treated with 200 μg/mL bLF, then infected with DENV-2 at MOI of 5, and analyzed by infectious center assay; ( B ) The infection rate equals 100 × (plaque number/cell number) (* p
    Fitc Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenotypic and functional characterization of the IL-33-induced Breg-like cells (Breg IL-33 ) in the blood. A–C. Surface phenotype of Breg IL-33 : The IL-33-induced IL-10 producing B cells expressed high surface IgM (A), CD1d and CD25 (B), but down-regulated CD23 (FceRII) (B, C). Data shown in the table under (B) were results showing the Mean MFI (±SD) values calculated from results of 5 repeated experiments, as ratio of the IL-10 − B, and IL-10 + B, cell subset over the total B cells gated. Statistical analysis: p values (Student t test). D–F. In vitro Breg suppression assays: Immunosuppressive effects of the IL-33-induced Breg-like cells on B effector (Beff) cell proliferation (D), division (E), and its IL-10 dependency (F). D. CD23 + B cells (Beff, 10 5 ) were cultured in the presence of anti-CD40 (2.5 μg/ml), with titrating (as indicated) doses of CD23 − B cells (Breg IL-33 ) purified from the IL-33-injected mice. Cell proliferation was determined by thymidine incorporation at 48 h s. E. Breg IL-33 (CD23 - ) purified from hIL-33-injected WT mice (Wk-2) were primed with anti-CD40 (2.5 μg/ml) for 5 h before being added to CFSE-labelled CD23 + Beff cells. Cell division (CFSE dilution) was determined by flow cytometry at day 5 of culture in the presence or absence of LPS (0.5 μg/ml) or anti-IgM (5 μg/ml). F. IL-10 levels in the culture supernatants were quantified by ELISA (BD Biosciences).

    Journal: Journal of Autoimmunity

    Article Title: IL-10-producing regulatory B cells induced by IL-33 (BregIL-33) effectively attenuate mucosal inflammatory responses in the gut

    doi: 10.1016/j.jaut.2014.01.032

    Figure Lengend Snippet: Phenotypic and functional characterization of the IL-33-induced Breg-like cells (Breg IL-33 ) in the blood. A–C. Surface phenotype of Breg IL-33 : The IL-33-induced IL-10 producing B cells expressed high surface IgM (A), CD1d and CD25 (B), but down-regulated CD23 (FceRII) (B, C). Data shown in the table under (B) were results showing the Mean MFI (±SD) values calculated from results of 5 repeated experiments, as ratio of the IL-10 − B, and IL-10 + B, cell subset over the total B cells gated. Statistical analysis: p values (Student t test). D–F. In vitro Breg suppression assays: Immunosuppressive effects of the IL-33-induced Breg-like cells on B effector (Beff) cell proliferation (D), division (E), and its IL-10 dependency (F). D. CD23 + B cells (Beff, 10 5 ) were cultured in the presence of anti-CD40 (2.5 μg/ml), with titrating (as indicated) doses of CD23 − B cells (Breg IL-33 ) purified from the IL-33-injected mice. Cell proliferation was determined by thymidine incorporation at 48 h s. E. Breg IL-33 (CD23 - ) purified from hIL-33-injected WT mice (Wk-2) were primed with anti-CD40 (2.5 μg/ml) for 5 h before being added to CFSE-labelled CD23 + Beff cells. Cell division (CFSE dilution) was determined by flow cytometry at day 5 of culture in the presence or absence of LPS (0.5 μg/ml) or anti-IgM (5 μg/ml). F. IL-10 levels in the culture supernatants were quantified by ELISA (BD Biosciences).

    Article Snippet: Respectively, fixed numbers (105 ) of B responder cells (CD19+ CD23+ ) were cultured, in the presence or absence of anti-CD40 (2.5 μg/ml, Enzo Life Sciences, UK), anti-IgM (5 μg/ml, Thermo Scientific, UK) or LPS (0.5 μg/ml, Sigma–Aldrich, UK), with titrated doses of BregIL-33 (CD19+ CD23− ) isolated from IL-33-treated WT or IL-10−/− mice as described above.

    Techniques: Functional Assay, In Vitro, Cell Culture, Purification, Injection, Mouse Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Plasmodium falciparum EBA175RIII–V construct and expressed antigen. A schematic representation of the 1620 bp region of EBA175RIII–V cloned into the pLEA2 expression vector, which contains the nucleotide sequence of a hexahistidine tag inframe of the multiple cloning site ( a ). The culture supernatant (1) containing the secreted protein as well as the purified protein (2) was analysed by SDS-PAGE followed by coomassie staining ( b ) and a western blot probed with penta-His mouse monoclonal antibody (IgG1) ( c )

    Journal: Malaria Journal

    Article Title: Assessment of the quality and quantity of naturally induced antibody responses to EBA175RIII–V in Ghanaian children living in two communities with varying malaria transmission patterns

    doi: 10.1186/s12936-017-2167-3

    Figure Lengend Snippet: Plasmodium falciparum EBA175RIII–V construct and expressed antigen. A schematic representation of the 1620 bp region of EBA175RIII–V cloned into the pLEA2 expression vector, which contains the nucleotide sequence of a hexahistidine tag inframe of the multiple cloning site ( a ). The culture supernatant (1) containing the secreted protein as well as the purified protein (2) was analysed by SDS-PAGE followed by coomassie staining ( b ) and a western blot probed with penta-His mouse monoclonal antibody (IgG1) ( c )

    Article Snippet: The western blot was probed with penta-His mouse IgG1 monoclonal antibodies (Thermo Scientific, USA) followed by alkaline phosphatase conjugated goat anti mouse IgG (H + L) secondary antibodies (Thermo Scientific, USA).

    Techniques: Construct, Clone Assay, Expressing, Plasmid Preparation, Sequencing, Purification, SDS Page, Staining, Western Blot

    Characterization of cytophilic antibody responses. IgG1 ( a ) and IgG3 ( b ) antibody concentrations to EBA175RIII–V Ll in asymptomatic children from Obom and Abura measured in the peak malaria season (July). Processes similar to that described in Fig. 3 were used to determine the concentrations and avidity of IgG1 and IgG3, the only difference was that goat anti-human IgG1 and goat anti-human IgG3 secondary antibodies were used in place of the goat anti-human IgG. The graphs represent the median antibody concentrations with interquartile range as error bars

    Journal: Malaria Journal

    Article Title: Assessment of the quality and quantity of naturally induced antibody responses to EBA175RIII–V in Ghanaian children living in two communities with varying malaria transmission patterns

    doi: 10.1186/s12936-017-2167-3

    Figure Lengend Snippet: Characterization of cytophilic antibody responses. IgG1 ( a ) and IgG3 ( b ) antibody concentrations to EBA175RIII–V Ll in asymptomatic children from Obom and Abura measured in the peak malaria season (July). Processes similar to that described in Fig. 3 were used to determine the concentrations and avidity of IgG1 and IgG3, the only difference was that goat anti-human IgG1 and goat anti-human IgG3 secondary antibodies were used in place of the goat anti-human IgG. The graphs represent the median antibody concentrations with interquartile range as error bars

    Article Snippet: The western blot was probed with penta-His mouse IgG1 monoclonal antibodies (Thermo Scientific, USA) followed by alkaline phosphatase conjugated goat anti mouse IgG (H + L) secondary antibodies (Thermo Scientific, USA).

    Techniques:

    Characterization of IgG responses against EBA175RIII–V Ll . Antibody concentrations (ng/ml) of plasma obtained from the enrolled children from Obom and Abura ( a ) diluted 1:200 was determined using indirect ELISA and a EBA175RIII–V Ll as the antigen coated onto the ELISA plate and goat anti-human IgG used as the secondary antibody. The relative avidities of these same plasma samples were determined using a modified indirect ELISA assay where an incubation of the bound plasma samples obtained from children Obom and Abura ( b ) were treated with sodium thiocyanide is incorporated into the protocol. Plasma samples were obtained from whole blood collected from the children during the months of July 2015, October 2015 and February 2016. Data in the graphs are represented as the median with the interquartile range

    Journal: Malaria Journal

    Article Title: Assessment of the quality and quantity of naturally induced antibody responses to EBA175RIII–V in Ghanaian children living in two communities with varying malaria transmission patterns

    doi: 10.1186/s12936-017-2167-3

    Figure Lengend Snippet: Characterization of IgG responses against EBA175RIII–V Ll . Antibody concentrations (ng/ml) of plasma obtained from the enrolled children from Obom and Abura ( a ) diluted 1:200 was determined using indirect ELISA and a EBA175RIII–V Ll as the antigen coated onto the ELISA plate and goat anti-human IgG used as the secondary antibody. The relative avidities of these same plasma samples were determined using a modified indirect ELISA assay where an incubation of the bound plasma samples obtained from children Obom and Abura ( b ) were treated with sodium thiocyanide is incorporated into the protocol. Plasma samples were obtained from whole blood collected from the children during the months of July 2015, October 2015 and February 2016. Data in the graphs are represented as the median with the interquartile range

    Article Snippet: The western blot was probed with penta-His mouse IgG1 monoclonal antibodies (Thermo Scientific, USA) followed by alkaline phosphatase conjugated goat anti mouse IgG (H + L) secondary antibodies (Thermo Scientific, USA).

    Techniques: Indirect ELISA, Enzyme-linked Immunosorbent Assay, Modification, Incubation

    Confocal microscopy of cells labeled with anti–LDL receptor Ab. ( a – f ) EBV-lymphocytes were incubated with rabbit anti–LDL receptor (red) at 4°C, washed at 4°C, and either directly permeabilized ( a – c ) or incubated for 10 minutes at 37°C before permeabilization to allow internalization of LDL receptor/Ab complexes ( d – f ). Permeabilized cells were then incubated with mouse anti–α-adaptin (AP2), washed, and incubated with Alexa 568–conjugated goat anti-rabbit IgG (LDL receptors, red) and Alexa 488–conjugated goat anti-mouse IgG (AP2, green). Nuclei were stained with DAPI. The plates shown are an overlay of red and green images. The bar represents 5.0 μm. ( a and d ) Control cells; ( b and e ) cells from proband 1.1; ( c and f ) cells from proband 1.1 expressing viral c-myc-ARH. ( g – l ) EBV-lymphocytes ( g – i ) or cultured skin fibroblasts ( j – l ) from three different control subjects were incubated with anti–LDL receptor Ab at 4°C, permeabilized, and then incubated with anti–α-adaptin Ab (AP2) as described for a – f above. The bars represent 5.0 μm (in g for g – i , and in j for j – l ).

    Journal: The Journal of Clinical Investigation

    Article Title: Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1

    doi: 10.1172/JCI16445

    Figure Lengend Snippet: Confocal microscopy of cells labeled with anti–LDL receptor Ab. ( a – f ) EBV-lymphocytes were incubated with rabbit anti–LDL receptor (red) at 4°C, washed at 4°C, and either directly permeabilized ( a – c ) or incubated for 10 minutes at 37°C before permeabilization to allow internalization of LDL receptor/Ab complexes ( d – f ). Permeabilized cells were then incubated with mouse anti–α-adaptin (AP2), washed, and incubated with Alexa 568–conjugated goat anti-rabbit IgG (LDL receptors, red) and Alexa 488–conjugated goat anti-mouse IgG (AP2, green). Nuclei were stained with DAPI. The plates shown are an overlay of red and green images. The bar represents 5.0 μm. ( a and d ) Control cells; ( b and e ) cells from proband 1.1; ( c and f ) cells from proband 1.1 expressing viral c-myc-ARH. ( g – l ) EBV-lymphocytes ( g – i ) or cultured skin fibroblasts ( j – l ) from three different control subjects were incubated with anti–LDL receptor Ab at 4°C, permeabilized, and then incubated with anti–α-adaptin Ab (AP2) as described for a – f above. The bars represent 5.0 μm (in g for g – i , and in j for j – l ).

    Article Snippet: Cells were fixed in 4% (wt/vol) paraformaldehyde, permeabilized in PBS containing 0.1% Triton X-100 and 10 mM glycine, incubated sequentially for 1 hour at ambient temperature with mouse monoclonal anti–α-adaptin Ab (Santa Cruz Biotechnology Inc.; diluted 1/100), Alexa 568–conjugated goat anti-rabbit IgG (highly cross-absorbed; Molecular Probes Europe BV, Leiden, The Netherlands; diluted 1/100), and Alexa 488–conjugated goat anti-mouse IgG (highly cross-absorbed; Molecular Probes, diluted 1/100), and then mounted on slides with VECTASHIELD plus 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) (Vector Laboratories, Peterborough, United Kingdom).

    Techniques: Confocal Microscopy, Labeling, Incubation, Staining, Expressing, Cell Culture

    Role of DC-SIGN in the anti-DENV2 activity of bLF. The THP-1 cells were stimulated with recombinant IL-4, GM-CSF, and TNF-α according to a previous report [ 19 ], and dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expression was subsequently measured by flow cytometry using mouse anti-human DC-SIGN antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) antibodies ( A ) The DC-SIGN-expressing and non-expessing THP-1 cells were treated with 200 μg/mL bLF, then infected with DENV-2 at MOI of 5, and analyzed by infectious center assay; ( B ) The infection rate equals 100 × (plaque number/cell number) (* p

    Journal: International Journal of Molecular Sciences

    Article Title: Bovine Lactoferrin Inhibits Dengue Virus Infectivity by Interacting with Heparan Sulfate, Low-Density Lipoprotein Receptor, and DC-SIGN

    doi: 10.3390/ijms18091957

    Figure Lengend Snippet: Role of DC-SIGN in the anti-DENV2 activity of bLF. The THP-1 cells were stimulated with recombinant IL-4, GM-CSF, and TNF-α according to a previous report [ 19 ], and dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expression was subsequently measured by flow cytometry using mouse anti-human DC-SIGN antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) antibodies ( A ) The DC-SIGN-expressing and non-expessing THP-1 cells were treated with 200 μg/mL bLF, then infected with DENV-2 at MOI of 5, and analyzed by infectious center assay; ( B ) The infection rate equals 100 × (plaque number/cell number) (* p

    Article Snippet: After seven days, the cells were fixed and stained with mouse anti-human DC-SIGN (AbD Serotec, Oxford, UK) and FITC-conjugated goat anti-mouse IgG (Invitrogen, Grand Island, NY USA).

    Techniques: Activity Assay, Recombinant, Expressing, Flow Cytometry, Cytometry, Infection