goat anti mouse igg Millipore Search Results


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  • 99
    Millipore anti mouse igg whole molecule peroxidase antibody
    Binding of <t>IgG</t> and <t>C3b</t> with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.
    Anti Mouse Igg Whole Molecule Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti rabbit igg whole molecule fitc antibody
    Immunohistochemical studies to detect deposition of aβ2GPI and NHIgG in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman <t>IgG</t> antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with <t>FITC-conjugated</t> antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.
    Anti Rabbit Igg Whole Molecule Fitc Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1508 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg fab specific peroxidase antibody
    Antibody conjugated nanoshells bound to cells when the appropriate antigen was present. SK-BR-3 cells were incubated with PEG-coated nanoshells (a, d), <t>anti-IgG</t> conjugated nanoshells (b, e), and anti-HER2 conjugated nanoshells (c, f). Following laser exposure, a region of cell death corresponding to the laser spot resulted in groups incubated with anti-HER2 conjugated nanoshells (c). Cells incubated with PEGylated or anti-IgG conjugated nanoshells continued to live. Silver staining (d–f) showed maximal binding of anti-HER2 conjugated nanoshells to the SK-BR-3 cells (f). Laser spot is 1.5 mm wide. Abbreviations: HER2, epidermal growth factor receptor; PEG, polyethylene glycol.
    Anti Mouse Igg Fab Specific Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg whole molecule fitc antibody
    The translation of a soluble form of ELR1 from ELR1-IN. Three approaches were used to confirm the expression of the predicted sELR1. ( A ) The recombinant sELR1 was expressed as a fusion protein with an HA tag at the C-terminus in 293T cells, and its presence in the cell lysate or in the culture medium was examined by Western blot using an anti-HA antibody. β-actin was applied as the internal control protein. (B) Live cell imaging analysis of ELR1 and sELR1 expression in HEK 293T cells. These two forms of receptor were expressed as proteins with a GFP fused at the N-terminus of each. The three images of sELR1 were enhanced at a higher level than ELR1. (C) Cellular location of ELR1 and sELR-1. HA-tagged ELR1 and sELR1 were expressed in 293T cells for 72 h. Cells were then examined by confocal microscopy after being fixed, stained with DAPI for the nucleus (blue) and DII for the cell membrane (red) and reacted with an anti-HA antibody and a <t>FITC-labeled</t> anti-mouse <t>IgG</t> (green). Appropriate IgG isotype controls were included to rule out non-specific binding.
    Anti Mouse Igg Whole Molecule Fitc Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti mouse igg whole molecule alkaline phosphatase antibody
    MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane. The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with <t>Flag-DP1</t> were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “ Materials and Methods ”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse <t>IgG</t> and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.
    Anti Mouse Igg Whole Molecule Alkaline Phosphatase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti goat igg
    <t>IgG,</t> <t>IgM,</t> and complement components C3, C4 and FB deposition on N . gonorrhoeae ( Ng) F62 that have incorporated each indicated sialic acid analog into lipooligosaccharide (LOS). Ng F62 ΔlgtD was grown in media alone (open bars), or media supplemented with 20 μg/ml (~30 μM) of each of the indicated CMP-Sias (NulOs). Bacteria were incubated with either 3.3% or 10% normal human serum (NHS) at 37°C for 10 min and IgG and IgM binding, and C3, C4 and factor B (FB) deposited on bacteria were measured by whole cell ELISA. Data with 3.3% and 10% NHS is shown using hatched and solid bars, respectively. mAb 2-8C-4-1 [ 62 ] that detects the Neisserial lipoprotein H.8 was similar across all groups and confirmed similar capture of bacteria in all wells (bottom right graph). Measurement of gonococcal H.8 lipoprotein antigen was performed to assess similarity of bacterial capture across microtiter wells. The mean (SD) of three observations is shown. Significance of differences in Ig or complement component binding/deposition using each analog vs, Neu5Ac is indicated for results that used 10% NHS only (for simplicity): *, P
    Anti Goat Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 785 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti mouse igg fc specific peroxidase antibody
    Dynamic interplay between O -GlcNAcylation and phosphorylation on Lsp1 in B cells after anti-IgM stimulation . ( a ) Mass spectrometric results revealing 12 mapped phosphorylation sites and 1 O -GlcNAcylation site on Lsp1. ( b ) Mapping of the O -GlcNAcylation site 208-KSQPTLPISTIDER-221 of Lsp1 using ETD fragmentation during MS/MS analysis. The ions, c 1 + (146.3 Da), c 2 + (436.3 Da), z+1 12 + (1353.5 Da), z+1 13 + (1643.8 Da) suggest that S209 is O -GlcNAcylated. ( c ) Lysates prepared from Ramos B cells overexpressing the vector control, Flag-EGFP-tagged WT or S209A Lsp1 were subjected to a pull-down assay using sWGA agarose beads, followed by <t>immunoblotting</t> (IB) with an anti-Flag antibody. ( d ) Lysates from 293T cells ectopically expressing OGT and either vector, Flag-EGFP-tagged WT or S209A Lsp1, were used for immunoprecipitation (IP) with anti-Flag, followed by IB with the indicated antibodies. ( e ) IB showing the levels of Lsp1, S243 phosphorylated Lsp1 and O -GlcNAcylated Lsp1 in anti-IgM (10 μg ml −1 ) stimulated mouse splenic B cells at indicated time points in an IP assay with control rabbit <t>IgG</t> (rIgG) or anti-Lsp1-specific antibody crosslinked to protein A agarose. The percentage of O -GlcNAcylated Lsp1 is indicated. ( f ) Sorted EGFP + Ramos B cells overexpressing Flag-EGFP-tagged WT or S209A Lsp1 were stimulated with anti-human IgM (25 μg ml −1 ) for 30 min, followed by IB analysis of Lsp1 S243 phosphorylation. One representative experiment out of three is shown. The relative levels of Lsp1 with phosphorylation at S243 were indicated. Results represent the mean±s.e.m. ( n =3). *** P
    Anti Mouse Igg Fc Specific Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore goat anti mouse igg
    Anti-lysozyme <t>IgG</t> response in high-responder H-2 k/b F 1 nontransgenic and TLK transgenic mice. ( A ) Mice were immunized i.p. with 100 μg <t>HEL</t> in RIBI adjuvant. ( B ) Mice were immunized i.p. with 25 μg HEL-cGG in RIBI adjuvant. In both cases, day 35 data represent secondary responses 7 d after boosting with HEL/RIBI or HEL-cGG/ RIBI, respectively. HEL binding IgG was measured by ELISA in serum of individual mice ( dots ), and geometric means are shown in bars.
    Goat Anti Mouse Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 3557 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore duolink in situ pla probe anti mouse minus
    TrkA and APP interaction in septal primary neurons measured by proximity ligation assay. (A) Confocal microscopy analysis of double-staining of APP (rabbit APP-CT A8717, red) and Trk (mouse Trk B3, green) in primary septal neurons showing co-localization of APP and TrkA. (B) The TrkA/APP complex was visualized by <t>PLA,</t> which generates red dots when the two proteins are in close proximity. Mouse and rabbit anti TrkA-CT (mouse Trk B3 and rabbit TrkA ab7261) and anti APP-CT (rabbit APP-CT A8717 and mouse APP clone C1-6.1) were used as primary antibodies and anti-mouse <t>MINUS</t> and anti-rabbit PLUS were used as secondary antibodies. Red fluorescent dot represents single interaction between TrkA and APP. (C) In addition to PLA (red dots), rat primary septal neurons were immunostained with goat anti ChAt (green) and with DAPI for nuclei (blue). (D) PLA assay using mouse anti APP (C1-6.1) and rabbit anti TrkB (sc-119) or TrkC (sc-117).
    Duolink In Situ Pla Probe Anti Mouse Minus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 374 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore anti mouse igg
    SIM1 measures degranulation from natural killer (NK) and CD8 T cells . (A) Percent degranulating cells were measured using anti-CD107a antibodies SIM1, H4A3, and LS-C8580, with or without YAC-1 target cells as a stimulus. One representative experiment of four is shown (B) . Staining of the <t>BWZ.CD107a-FLAG</t> cell line with SIM1, H4A3, LS-C8580, or ID48 antibody clones. BWZ.36 cells transfected with irrelevant FLAG-tagged antigen (BWZ.NKR-P1G-FLAG) was included as a negative control, (C) enriched NK cells were stimulated with plate bound anti-NKp46, anti-NKR-P1A, or mouse IgG1 as isotype control, and SIM1 was used to measure percentage degranulating cells. NKp46 + (Wen23) or NKR-P1A bright (3.2.3) cells were gated on. (D) TCR + cells were sorted (purity 99%) and cultured in IL-2 for 2–3 days. The cells were then stimulated with plate bound anti-CD3 and anti-CD2 or mouse IgM isotype control for anti-CD3. The lower panel shows stimulation with anti-CD3 and anti-CD2 and staining with a SIM1 isotype control (FITC hamster <t>IgG</t> polyclonal). T cells were sorted using R73-Alexa647 and 3.2.3-PB antibodies, gating on R73 + /3.2.3 − cells. Data shown are from one representative experiment of three separate independent experiments.
    Anti Mouse Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 4701 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore anti mouse igg gamma chain specific peroxidase antibody
    SIM1 measures degranulation from natural killer (NK) and CD8 T cells . (A) Percent degranulating cells were measured using anti-CD107a antibodies SIM1, H4A3, and LS-C8580, with or without YAC-1 target cells as a stimulus. One representative experiment of four is shown (B) . Staining of the <t>BWZ.CD107a-FLAG</t> cell line with SIM1, H4A3, LS-C8580, or ID48 antibody clones. BWZ.36 cells transfected with irrelevant FLAG-tagged antigen (BWZ.NKR-P1G-FLAG) was included as a negative control, (C) enriched NK cells were stimulated with plate bound anti-NKp46, anti-NKR-P1A, or mouse IgG1 as isotype control, and SIM1 was used to measure percentage degranulating cells. NKp46 + (Wen23) or NKR-P1A bright (3.2.3) cells were gated on. (D) TCR + cells were sorted (purity 99%) and cultured in IL-2 for 2–3 days. The cells were then stimulated with plate bound anti-CD3 and anti-CD2 or mouse IgM isotype control for anti-CD3. The lower panel shows stimulation with anti-CD3 and anti-CD2 and staining with a SIM1 isotype control (FITC hamster <t>IgG</t> polyclonal). T cells were sorted using R73-Alexa647 and 3.2.3-PB antibodies, gating on R73 + /3.2.3 − cells. Data shown are from one representative experiment of three separate independent experiments.
    Anti Mouse Igg Gamma Chain Specific Peroxidase Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore secondary antibodies
    SIM1 measures degranulation from natural killer (NK) and CD8 T cells . (A) Percent degranulating cells were measured using anti-CD107a antibodies SIM1, H4A3, and LS-C8580, with or without YAC-1 target cells as a stimulus. One representative experiment of four is shown (B) . Staining of the <t>BWZ.CD107a-FLAG</t> cell line with SIM1, H4A3, LS-C8580, or ID48 antibody clones. BWZ.36 cells transfected with irrelevant FLAG-tagged antigen (BWZ.NKR-P1G-FLAG) was included as a negative control, (C) enriched NK cells were stimulated with plate bound anti-NKp46, anti-NKR-P1A, or mouse IgG1 as isotype control, and SIM1 was used to measure percentage degranulating cells. NKp46 + (Wen23) or NKR-P1A bright (3.2.3) cells were gated on. (D) TCR + cells were sorted (purity 99%) and cultured in IL-2 for 2–3 days. The cells were then stimulated with plate bound anti-CD3 and anti-CD2 or mouse IgM isotype control for anti-CD3. The lower panel shows stimulation with anti-CD3 and anti-CD2 and staining with a SIM1 isotype control (FITC hamster <t>IgG</t> polyclonal). T cells were sorted using R73-Alexa647 and 3.2.3-PB antibodies, gating on R73 + /3.2.3 − cells. Data shown are from one representative experiment of three separate independent experiments.
    Secondary Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2204 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore anti rabbit igg whole molecule biotin antibody
    SIM1 measures degranulation from natural killer (NK) and CD8 T cells . (A) Percent degranulating cells were measured using anti-CD107a antibodies SIM1, H4A3, and LS-C8580, with or without YAC-1 target cells as a stimulus. One representative experiment of four is shown (B) . Staining of the <t>BWZ.CD107a-FLAG</t> cell line with SIM1, H4A3, LS-C8580, or ID48 antibody clones. BWZ.36 cells transfected with irrelevant FLAG-tagged antigen (BWZ.NKR-P1G-FLAG) was included as a negative control, (C) enriched NK cells were stimulated with plate bound anti-NKp46, anti-NKR-P1A, or mouse IgG1 as isotype control, and SIM1 was used to measure percentage degranulating cells. NKp46 + (Wen23) or NKR-P1A bright (3.2.3) cells were gated on. (D) TCR + cells were sorted (purity 99%) and cultured in IL-2 for 2–3 days. The cells were then stimulated with plate bound anti-CD3 and anti-CD2 or mouse IgM isotype control for anti-CD3. The lower panel shows stimulation with anti-CD3 and anti-CD2 and staining with a SIM1 isotype control (FITC hamster <t>IgG</t> polyclonal). T cells were sorted using R73-Alexa647 and 3.2.3-PB antibodies, gating on R73 + /3.2.3 − cells. Data shown are from one representative experiment of three separate independent experiments.
    Anti Rabbit Igg Whole Molecule Biotin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 341 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore hrp conjugated goat anti mouse igg
    SDS-PAGE and Western blot analysis of purified recombinant NS1 protein expressed in E . coli . Lane M, molecular mass markers; lane 1, SDS-PAGE analysis of purified recombinant NS1 protein; Lane 2, the recombinant NS1 protein was analyzed by western blotting using SPF chicken serum for the negative control. Lane 3, western blotting of the purified recombinant NS1 protein, with chicken anti-AIV antibody and horseradish peroxidase <t>(HRP)-labeled</t> sheep anti-chicken <t>IgG</t> H+L as the first and second antibody, respectively. The arrowhead indicates the position of recombinant NS1 protein (approximate molecular mass of 30 kDa).
    Hrp Conjugated Goat Anti Mouse Igg, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore anti mouse igg fab specific fitc antibody
    SDS-PAGE and Western blot analysis of purified recombinant NS1 protein expressed in E . coli . Lane M, molecular mass markers; lane 1, SDS-PAGE analysis of purified recombinant NS1 protein; Lane 2, the recombinant NS1 protein was analyzed by western blotting using SPF chicken serum for the negative control. Lane 3, western blotting of the purified recombinant NS1 protein, with chicken anti-AIV antibody and horseradish peroxidase <t>(HRP)-labeled</t> sheep anti-chicken <t>IgG</t> H+L as the first and second antibody, respectively. The arrowhead indicates the position of recombinant NS1 protein (approximate molecular mass of 30 kDa).
    Anti Mouse Igg Fab Specific Fitc Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Millipore anti mouse igg whole molecule antibody
    A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit <t>IgG</t> ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and <t>brefeldin</t> A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.
    Anti Mouse Igg Whole Molecule Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 473 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore anti mouse igg fab specific biotin antibody
    A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit <t>IgG</t> ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and <t>brefeldin</t> A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.
    Anti Mouse Igg Fab Specific Biotin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore duolink in situ pla probe anti rabbit minus
    Assembly is restricted between the members of the TREK/TRAAK subfamily. ( A ) Phylogenetic tree of human K 2P subunits. The tree was constructed with a multiple alignment using fast Fourier transform (MAFFT) program. ( B ) <t>Duolink</t> in situ <t>PLA</t> between TREK1 and other K 2P subunits representative of the different subfamilies. HA-TREK1 was coexpressed in MDCK cells with Myc-tagged subunits. Percentage of positive cells corresponds to the number of PLA-positive cells relative to the number of transfected cells. Number of independent experiments is given in parenthesis and corresponds to > 300 cells analyzed per condition. Data were analyzed by using Mann–Whitney test: * P
    Duolink In Situ Pla Probe Anti Rabbit Minus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore duolink in situ pla probe anti mouse plus
    Indirect <t>PLA</t> in cells. Cells co-expressing GPCR protomer A and GPCR protomer B are incubated with anti-mouse antibody selective for protomer A and anti-rabbit antibody selective for protomer B, followed by incubation with species-specific PLA probes <t>PLUS</t> and MINUS. Ligation of the oligonucleotide sequences creates a rolling circle template that is amplified by polymerase in the presence of fluorescent oligonucleotides and can be detected as red dots by confocal microscopy with the nuclei stained in blue by DAPI.
    Duolink In Situ Pla Probe Anti Mouse Plus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Binding of IgG and C3b with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.

    Journal: Frontiers in Immunology

    Article Title: Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation

    doi: 10.3389/fimmu.2020.00462

    Figure Lengend Snippet: Binding of IgG and C3b with encapsulated strain of Bacillus anthracis incubated at different pH buffers ranging from 2.4 to 7.4, supplemented with 10% normal human serum or 50 μg/ml of purified human IgG. (A) Immunoblot assay for the detection of purified IgG binding on encapsulated strain of B. anthracis . IgG binding was observed at pH closer to the isoelectric point of poly-γ-D-glutamate (pI 3.2). (B) Purified IgG was stable at different pH buffers as detected by immunoblot. (C) Immunoblot assay for the detection of human C3b binding on encapsulated strain of B. anthracis . C3b binding was also observed at pH closer to the isoelectric point of poly-γ-D-glutamate. (D) Normal human serum (NHS) was incubated at different pH buffers and C3 was observed to be stable at low pH as analyzed by immunoblot.

    Article Snippet: C3b was detected in bacterial lysates ran on a 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) without heating under non-reducing conditions (without adding 2-mercaptoethanol), transferred to nitrocellulose membrane (MDI), and probed with mouse anti-human C3b monoclonal antibodies (Thermo Fisher, Cat. No. MA1-70054) followed with horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (Sigma, Cat. No. A4416).

    Techniques: Binding Assay, Incubation, Purification

    Immunohistochemical studies to detect deposition of aβ2GPI and NHIgG in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman IgG antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with FITC-conjugated antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.

    Journal: Journal of autoimmunity

    Article Title: Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome

    doi: 10.1016/j.jaut.2016.04.008

    Figure Lengend Snippet: Immunohistochemical studies to detect deposition of aβ2GPI and NHIgG in placenta and fetal brain. Expression of β 2 GPI protein in placenta and fetal brain in control mice . aβ2GPI and NHIgG deposition in mouse tissue (placenta and fetal cortical brain) was detected using an antihuman IgG antibody labelled with Texas Red. A- detection of aβ 2 GPI in the placenta from mouse injected aβ 2 GPI. B- Detection of aβ 2 GPI in fetal cortical brain from a mouse injected with aβ 2 GPI. C- Detection of NHIgG in the placenta from a mouse injected with NHIgG. D- Detection of NHIgG in the fetal cortical brain in a mouse injected with NHIgG. Expression of β 2 GPI in placenta (E) and fetal brain (F) was detected with FITC-conjugated antibodies (green fluorescence). Diamidino-2-phenylindole (DAPI) was used for nuclear counterstains in all immunofluorescence studies. Data are representative of observations in 5–6 mice per group. 10 views per slide were analyzed in each experimental condition.

    Article Snippet: Bound antibodies were detected using a FITC-conjugated anti rabbit IgG (Sigma-Aldrich F9887, dilution 1/400).

    Techniques: Immunohistochemistry, Expressing, Mouse Assay, Injection, Fluorescence, Immunofluorescence

    Antibody conjugated nanoshells bound to cells when the appropriate antigen was present. SK-BR-3 cells were incubated with PEG-coated nanoshells (a, d), anti-IgG conjugated nanoshells (b, e), and anti-HER2 conjugated nanoshells (c, f). Following laser exposure, a region of cell death corresponding to the laser spot resulted in groups incubated with anti-HER2 conjugated nanoshells (c). Cells incubated with PEGylated or anti-IgG conjugated nanoshells continued to live. Silver staining (d–f) showed maximal binding of anti-HER2 conjugated nanoshells to the SK-BR-3 cells (f). Laser spot is 1.5 mm wide. Abbreviations: HER2, epidermal growth factor receptor; PEG, polyethylene glycol.

    Journal: International Journal of Nanomedicine

    Article Title: Immunonanoshells for targeted photothermal ablation of tumor cells

    doi:

    Figure Lengend Snippet: Antibody conjugated nanoshells bound to cells when the appropriate antigen was present. SK-BR-3 cells were incubated with PEG-coated nanoshells (a, d), anti-IgG conjugated nanoshells (b, e), and anti-HER2 conjugated nanoshells (c, f). Following laser exposure, a region of cell death corresponding to the laser spot resulted in groups incubated with anti-HER2 conjugated nanoshells (c). Cells incubated with PEGylated or anti-IgG conjugated nanoshells continued to live. Silver staining (d–f) showed maximal binding of anti-HER2 conjugated nanoshells to the SK-BR-3 cells (f). Laser spot is 1.5 mm wide. Abbreviations: HER2, epidermal growth factor receptor; PEG, polyethylene glycol.

    Article Snippet: Quantification of the number of antibodies per nanoshell (ELISA) Immunonanoshells were incubated with horseradish peroxidase-labeled (HRP) anti-mouse IgG (Sigma-Aldrich, A3682) for 1 hour.

    Techniques: Incubation, Silver Staining, Binding Assay

    The translation of a soluble form of ELR1 from ELR1-IN. Three approaches were used to confirm the expression of the predicted sELR1. ( A ) The recombinant sELR1 was expressed as a fusion protein with an HA tag at the C-terminus in 293T cells, and its presence in the cell lysate or in the culture medium was examined by Western blot using an anti-HA antibody. β-actin was applied as the internal control protein. (B) Live cell imaging analysis of ELR1 and sELR1 expression in HEK 293T cells. These two forms of receptor were expressed as proteins with a GFP fused at the N-terminus of each. The three images of sELR1 were enhanced at a higher level than ELR1. (C) Cellular location of ELR1 and sELR-1. HA-tagged ELR1 and sELR1 were expressed in 293T cells for 72 h. Cells were then examined by confocal microscopy after being fixed, stained with DAPI for the nucleus (blue) and DII for the cell membrane (red) and reacted with an anti-HA antibody and a FITC-labeled anti-mouse IgG (green). Appropriate IgG isotype controls were included to rule out non-specific binding.

    Journal: PLoS ONE

    Article Title: The Soluble Form of the EIAV Receptor Encoded by an Alternative Splicing Variant Inhibits EIAV Infection of Target Cells

    doi: 10.1371/journal.pone.0079299

    Figure Lengend Snippet: The translation of a soluble form of ELR1 from ELR1-IN. Three approaches were used to confirm the expression of the predicted sELR1. ( A ) The recombinant sELR1 was expressed as a fusion protein with an HA tag at the C-terminus in 293T cells, and its presence in the cell lysate or in the culture medium was examined by Western blot using an anti-HA antibody. β-actin was applied as the internal control protein. (B) Live cell imaging analysis of ELR1 and sELR1 expression in HEK 293T cells. These two forms of receptor were expressed as proteins with a GFP fused at the N-terminus of each. The three images of sELR1 were enhanced at a higher level than ELR1. (C) Cellular location of ELR1 and sELR-1. HA-tagged ELR1 and sELR1 were expressed in 293T cells for 72 h. Cells were then examined by confocal microscopy after being fixed, stained with DAPI for the nucleus (blue) and DII for the cell membrane (red) and reacted with an anti-HA antibody and a FITC-labeled anti-mouse IgG (green). Appropriate IgG isotype controls were included to rule out non-specific binding.

    Article Snippet: Cells were blocked with PBS/FISH GELATIN (5%) for 2 h, reacted with anti-HA monoclonal Ab for 1 h and then incubated with an anti-mouse IgG (whole molecule)-fluorescein isothiocyanate (FITC)-labeled antibody (F2012, Sigma).

    Techniques: Expressing, Recombinant, Western Blot, Live Cell Imaging, Confocal Microscopy, Staining, Labeling, Binding Assay

    MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane. The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with Flag-DP1 were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “ Materials and Methods ”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.

    Journal: PLoS ONE

    Article Title: Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor

    doi: 10.1371/journal.pone.0065767

    Figure Lengend Snippet: MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane. The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with Flag-DP1 were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “ Materials and Methods ”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.

    Article Snippet: Reagents Monoclonal anti-FLAG (M2) (cat. F3165), monoclonal anti-FLAG (M1) (cat. F3040), and goat alkaline phosphatase-conjugated anti-mouse IgG (cat. A3562) antibodies were from Sigma-Aldrich, MO.

    Techniques: Translocation Assay, Immunofluorescence, Confocal Microscopy, Transfection, Labeling

    IgG, IgM, and complement components C3, C4 and FB deposition on N . gonorrhoeae ( Ng) F62 that have incorporated each indicated sialic acid analog into lipooligosaccharide (LOS). Ng F62 ΔlgtD was grown in media alone (open bars), or media supplemented with 20 μg/ml (~30 μM) of each of the indicated CMP-Sias (NulOs). Bacteria were incubated with either 3.3% or 10% normal human serum (NHS) at 37°C for 10 min and IgG and IgM binding, and C3, C4 and factor B (FB) deposited on bacteria were measured by whole cell ELISA. Data with 3.3% and 10% NHS is shown using hatched and solid bars, respectively. mAb 2-8C-4-1 [ 62 ] that detects the Neisserial lipoprotein H.8 was similar across all groups and confirmed similar capture of bacteria in all wells (bottom right graph). Measurement of gonococcal H.8 lipoprotein antigen was performed to assess similarity of bacterial capture across microtiter wells. The mean (SD) of three observations is shown. Significance of differences in Ig or complement component binding/deposition using each analog vs, Neu5Ac is indicated for results that used 10% NHS only (for simplicity): *, P

    Journal: PLoS Pathogens

    Article Title: Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics

    doi: 10.1371/journal.ppat.1005290

    Figure Lengend Snippet: IgG, IgM, and complement components C3, C4 and FB deposition on N . gonorrhoeae ( Ng) F62 that have incorporated each indicated sialic acid analog into lipooligosaccharide (LOS). Ng F62 ΔlgtD was grown in media alone (open bars), or media supplemented with 20 μg/ml (~30 μM) of each of the indicated CMP-Sias (NulOs). Bacteria were incubated with either 3.3% or 10% normal human serum (NHS) at 37°C for 10 min and IgG and IgM binding, and C3, C4 and factor B (FB) deposited on bacteria were measured by whole cell ELISA. Data with 3.3% and 10% NHS is shown using hatched and solid bars, respectively. mAb 2-8C-4-1 [ 62 ] that detects the Neisserial lipoprotein H.8 was similar across all groups and confirmed similar capture of bacteria in all wells (bottom right graph). Measurement of gonococcal H.8 lipoprotein antigen was performed to assess similarity of bacterial capture across microtiter wells. The mean (SD) of three observations is shown. Significance of differences in Ig or complement component binding/deposition using each analog vs, Neu5Ac is indicated for results that used 10% NHS only (for simplicity): *, P

    Article Snippet: FITC conjugated anti-mouse IgG and anti-goat IgG, and alkaline phosphatase conjugated anti-mouse IgM, anti-mouse IgG and anti-goat IgG were all obtained from Sigma.

    Techniques: Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay

    The intracellular location of ORFV011 protein, ORFV059 protein and ORFV 011/ORFV059 chemeric-proteins in transfected OFTu cells . The OFTu cells were seeded in 6-well culture plates and un-transfected (Figure 2a) or transfected with pcDNA3.1(+) vector (Figure 2b), pcDNA3.1-ORFV011 plasmid (Figure 2c), pcDNA3.1-ORFV059 plasmid (Figure 2d) or pcDNA3.1-ORFV011/ORFV059 plasmid (Figure 2e) for 48 h. After 48 h post-transfection, the cells were fixed with 80% acetone for 10 min at -20°C, rehydrated in PBS, labeled with a rabbit anti-ORFV polyclonal antibody, and washed three times with PBS. FITC-conjugated goat anti-rabbit IgG (H + L) (1:500 dilution in PBS) was added to the OFTu cell mixtures for 30 min at room temperature, and the cells were washed and observed with a fluorescence microscopy. Magnification: 400 ×.

    Journal: Virology Journal

    Article Title: Orf virus DNA vaccines expressing ORFV 011 and ORFV 059 chimeric protein enhances immunogenicity

    doi: 10.1186/1743-422X-8-562

    Figure Lengend Snippet: The intracellular location of ORFV011 protein, ORFV059 protein and ORFV 011/ORFV059 chemeric-proteins in transfected OFTu cells . The OFTu cells were seeded in 6-well culture plates and un-transfected (Figure 2a) or transfected with pcDNA3.1(+) vector (Figure 2b), pcDNA3.1-ORFV011 plasmid (Figure 2c), pcDNA3.1-ORFV059 plasmid (Figure 2d) or pcDNA3.1-ORFV011/ORFV059 plasmid (Figure 2e) for 48 h. After 48 h post-transfection, the cells were fixed with 80% acetone for 10 min at -20°C, rehydrated in PBS, labeled with a rabbit anti-ORFV polyclonal antibody, and washed three times with PBS. FITC-conjugated goat anti-rabbit IgG (H + L) (1:500 dilution in PBS) was added to the OFTu cell mixtures for 30 min at room temperature, and the cells were washed and observed with a fluorescence microscopy. Magnification: 400 ×.

    Article Snippet: After a 1 h incubation, the plates were washed with PBS/0.1% Tween-20 and goat anti mouse IgG1, IgG2a (Sigma Chemical Co., SA Locus, Mo.) diluted to 1:1000 were used to detect IgG1 and IgG2a subtypes, respectively.

    Techniques: Transfection, Plasmid Preparation, Labeling, Fluorescence, Microscopy

    Group mean optical density ratios and standard errors for specific anti-ORFV-IgG antibody responses in the serum samples of immunized mice of different groups . The sera were collected from mice in groups (pcDNA3.1-ORFV011 group, pcDNA3.1-ORFV059 group, pcDNA3.1-ORFV011/ORFV059 group, pcDNA3.1-ORFV 011 plus pcDNA3.1-ORFV059 group, pcDNA3.1(+) group and PBS group) at weeks 0, 1, 2, 3, 4, 5, 6, 7, 8 and were inactivated at 56° for 30 min. The ORFV-specific antibody responses were determined using an indirect ELISA, with the purified ORFV as the coating antigen. As shown in Figure 3, the levels of anti-ORFV-IgG antibodies were developed in different DNA vaccine plasmid- immunized group. One week after the final boost immunization, the level of anti-ORFV-IgG antibodies was higher in the pcDNA3.1-ORFV 011/ORFV 059 immunized group than any of the other groups tested (OD 450 value: 0.829 ± 0.014, ** P

    Journal: Virology Journal

    Article Title: Orf virus DNA vaccines expressing ORFV 011 and ORFV 059 chimeric protein enhances immunogenicity

    doi: 10.1186/1743-422X-8-562

    Figure Lengend Snippet: Group mean optical density ratios and standard errors for specific anti-ORFV-IgG antibody responses in the serum samples of immunized mice of different groups . The sera were collected from mice in groups (pcDNA3.1-ORFV011 group, pcDNA3.1-ORFV059 group, pcDNA3.1-ORFV011/ORFV059 group, pcDNA3.1-ORFV 011 plus pcDNA3.1-ORFV059 group, pcDNA3.1(+) group and PBS group) at weeks 0, 1, 2, 3, 4, 5, 6, 7, 8 and were inactivated at 56° for 30 min. The ORFV-specific antibody responses were determined using an indirect ELISA, with the purified ORFV as the coating antigen. As shown in Figure 3, the levels of anti-ORFV-IgG antibodies were developed in different DNA vaccine plasmid- immunized group. One week after the final boost immunization, the level of anti-ORFV-IgG antibodies was higher in the pcDNA3.1-ORFV 011/ORFV 059 immunized group than any of the other groups tested (OD 450 value: 0.829 ± 0.014, ** P

    Article Snippet: After a 1 h incubation, the plates were washed with PBS/0.1% Tween-20 and goat anti mouse IgG1, IgG2a (Sigma Chemical Co., SA Locus, Mo.) diluted to 1:1000 were used to detect IgG1 and IgG2a subtypes, respectively.

    Techniques: Mouse Assay, Indirect ELISA, Purification, Plasmid Preparation

    The subclasses IgG1 and IgG2a of anti-ORFV IgG antibodies in the serum samples of immunized mice of different groups . Sera were taken from immunized mice of different groups 1 week post the final boost immunization. The titration of ORFV-specific immunoglobulin (IgG) subclasses 1 and 2a was detected by ELISA. As shown in Figure 4, the assay of IgG2a and IgG1 antibodies performed at 1 week post the final boost immunization indicates a significant difference between vaccine plasmids groups (including pcDNA 3.1-ORFV011 group, pcDNA 3.1-ORFV059 group, pcDNA3.1-ORFV011 plus pcDNA3.1-ORFV059 group and pcDNA3.1-ORFV011/ORFV059 group) and control groups (including PBS and pcDNA3.1(+) group) (** P

    Journal: Virology Journal

    Article Title: Orf virus DNA vaccines expressing ORFV 011 and ORFV 059 chimeric protein enhances immunogenicity

    doi: 10.1186/1743-422X-8-562

    Figure Lengend Snippet: The subclasses IgG1 and IgG2a of anti-ORFV IgG antibodies in the serum samples of immunized mice of different groups . Sera were taken from immunized mice of different groups 1 week post the final boost immunization. The titration of ORFV-specific immunoglobulin (IgG) subclasses 1 and 2a was detected by ELISA. As shown in Figure 4, the assay of IgG2a and IgG1 antibodies performed at 1 week post the final boost immunization indicates a significant difference between vaccine plasmids groups (including pcDNA 3.1-ORFV011 group, pcDNA 3.1-ORFV059 group, pcDNA3.1-ORFV011 plus pcDNA3.1-ORFV059 group and pcDNA3.1-ORFV011/ORFV059 group) and control groups (including PBS and pcDNA3.1(+) group) (** P

    Article Snippet: After a 1 h incubation, the plates were washed with PBS/0.1% Tween-20 and goat anti mouse IgG1, IgG2a (Sigma Chemical Co., SA Locus, Mo.) diluted to 1:1000 were used to detect IgG1 and IgG2a subtypes, respectively.

    Techniques: Mouse Assay, Titration, Enzyme-linked Immunosorbent Assay

    Dynamic interplay between O -GlcNAcylation and phosphorylation on Lsp1 in B cells after anti-IgM stimulation . ( a ) Mass spectrometric results revealing 12 mapped phosphorylation sites and 1 O -GlcNAcylation site on Lsp1. ( b ) Mapping of the O -GlcNAcylation site 208-KSQPTLPISTIDER-221 of Lsp1 using ETD fragmentation during MS/MS analysis. The ions, c 1 + (146.3 Da), c 2 + (436.3 Da), z+1 12 + (1353.5 Da), z+1 13 + (1643.8 Da) suggest that S209 is O -GlcNAcylated. ( c ) Lysates prepared from Ramos B cells overexpressing the vector control, Flag-EGFP-tagged WT or S209A Lsp1 were subjected to a pull-down assay using sWGA agarose beads, followed by immunoblotting (IB) with an anti-Flag antibody. ( d ) Lysates from 293T cells ectopically expressing OGT and either vector, Flag-EGFP-tagged WT or S209A Lsp1, were used for immunoprecipitation (IP) with anti-Flag, followed by IB with the indicated antibodies. ( e ) IB showing the levels of Lsp1, S243 phosphorylated Lsp1 and O -GlcNAcylated Lsp1 in anti-IgM (10 μg ml −1 ) stimulated mouse splenic B cells at indicated time points in an IP assay with control rabbit IgG (rIgG) or anti-Lsp1-specific antibody crosslinked to protein A agarose. The percentage of O -GlcNAcylated Lsp1 is indicated. ( f ) Sorted EGFP + Ramos B cells overexpressing Flag-EGFP-tagged WT or S209A Lsp1 were stimulated with anti-human IgM (25 μg ml −1 ) for 30 min, followed by IB analysis of Lsp1 S243 phosphorylation. One representative experiment out of three is shown. The relative levels of Lsp1 with phosphorylation at S243 were indicated. Results represent the mean±s.e.m. ( n =3). *** P

    Journal: Nature Communications

    Article Title: Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells

    doi: 10.1038/ncomms12526

    Figure Lengend Snippet: Dynamic interplay between O -GlcNAcylation and phosphorylation on Lsp1 in B cells after anti-IgM stimulation . ( a ) Mass spectrometric results revealing 12 mapped phosphorylation sites and 1 O -GlcNAcylation site on Lsp1. ( b ) Mapping of the O -GlcNAcylation site 208-KSQPTLPISTIDER-221 of Lsp1 using ETD fragmentation during MS/MS analysis. The ions, c 1 + (146.3 Da), c 2 + (436.3 Da), z+1 12 + (1353.5 Da), z+1 13 + (1643.8 Da) suggest that S209 is O -GlcNAcylated. ( c ) Lysates prepared from Ramos B cells overexpressing the vector control, Flag-EGFP-tagged WT or S209A Lsp1 were subjected to a pull-down assay using sWGA agarose beads, followed by immunoblotting (IB) with an anti-Flag antibody. ( d ) Lysates from 293T cells ectopically expressing OGT and either vector, Flag-EGFP-tagged WT or S209A Lsp1, were used for immunoprecipitation (IP) with anti-Flag, followed by IB with the indicated antibodies. ( e ) IB showing the levels of Lsp1, S243 phosphorylated Lsp1 and O -GlcNAcylated Lsp1 in anti-IgM (10 μg ml −1 ) stimulated mouse splenic B cells at indicated time points in an IP assay with control rabbit IgG (rIgG) or anti-Lsp1-specific antibody crosslinked to protein A agarose. The percentage of O -GlcNAcylated Lsp1 is indicated. ( f ) Sorted EGFP + Ramos B cells overexpressing Flag-EGFP-tagged WT or S209A Lsp1 were stimulated with anti-human IgM (25 μg ml −1 ) for 30 min, followed by IB analysis of Lsp1 S243 phosphorylation. One representative experiment out of three is shown. The relative levels of Lsp1 with phosphorylation at S243 were indicated. Results represent the mean±s.e.m. ( n =3). *** P

    Article Snippet: Secondary antibodies used in immunoblotting are goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (Sigma, A2554, 1:5,000), goat anti-rabbit IgG HRP-conjugated antibody (Sigma, A0545, 1:5,000) and rabbit anti-goat IgG HRP-conjugated antibody (Sigma, A5420, 1:5,000).

    Techniques: Mass Spectrometry, Plasmid Preparation, Pull Down Assay, Expressing, Immunoprecipitation

    Phosphorylation of FG nucleoporins. (A) Extracts from vEC 9 - or mock-infected cells (4.5 h p.i.) were reacted with MAb 414 or IgG (control) in immunoprecipitation experiments (as described in Materials and Methods). Captured protein was treated with calf intestinal alkaline phosphatase before fractionation by PAGE and visualization by Western blot analysis (MAb 414). IgG immunoprecipitations from virus-infected and mock-infected cell lysates had identical patterns, and therefore the virus IgG control was not included in this figure. (B to D) Samples collected as described for panel A were fractionated on parallel gels by PAGE. (B) Band visualization was achieved by using Western blot analysis (MAb 414). (C) Band visualization was achieved by using the Pro-Q Diamond phosphoprotein stain. PO 4 Std, phosphoprotein reference standard. (D) Gel lanes in panel C corresponding with samples from vEC 9 -infected cells immunoprecipitated with MAb 414 (mAb414 IP; solid black line; lane 9 in panel C), mock-infected cell samples immunoprecipitated with IgG (IgG IP; solid gray line; lane 7 in panel C), or MAb 414 (dashed black line; lane 8 in panel C) were scanned and plotted. Asterisks denote bands from the mock-infected cells which cross-reacted with both antibodies (i.e., background).

    Journal: Journal of Virology

    Article Title: Leader-Induced Phosphorylation of Nucleoporins Correlates with Nuclear Trafficking Inhibition by Cardioviruses ▿

    doi: 10.1128/JVI.01752-08

    Figure Lengend Snippet: Phosphorylation of FG nucleoporins. (A) Extracts from vEC 9 - or mock-infected cells (4.5 h p.i.) were reacted with MAb 414 or IgG (control) in immunoprecipitation experiments (as described in Materials and Methods). Captured protein was treated with calf intestinal alkaline phosphatase before fractionation by PAGE and visualization by Western blot analysis (MAb 414). IgG immunoprecipitations from virus-infected and mock-infected cell lysates had identical patterns, and therefore the virus IgG control was not included in this figure. (B to D) Samples collected as described for panel A were fractionated on parallel gels by PAGE. (B) Band visualization was achieved by using Western blot analysis (MAb 414). (C) Band visualization was achieved by using the Pro-Q Diamond phosphoprotein stain. PO 4 Std, phosphoprotein reference standard. (D) Gel lanes in panel C corresponding with samples from vEC 9 -infected cells immunoprecipitated with MAb 414 (mAb414 IP; solid black line; lane 9 in panel C), mock-infected cell samples immunoprecipitated with IgG (IgG IP; solid gray line; lane 7 in panel C), or MAb 414 (dashed black line; lane 8 in panel C) were scanned and plotted. Asterisks denote bands from the mock-infected cells which cross-reacted with both antibodies (i.e., background).

    Article Snippet: Primary reagents against tubulin (murine MAb, SC-5286; Santa Cruz Biotech), eIF-4G (rabbit polyclonal, SC-11373; Santa Cruz Biotech), GFP (rabbit polyclonal, SC-8334; Santa Cruz Biotech), lamin A (murine MAb, ab8980; Abcam), and FG-containing nucleoporins (murine MAb 414; Covance) were commercial, as were appropriate horseradish peroxidase-conjugated secondary antibodies (A2554 and A0545; Sigma).

    Techniques: Infection, Immunoprecipitation, Fractionation, Polyacrylamide Gel Electrophoresis, Western Blot, Staining

    Anti-lysozyme IgG response in high-responder H-2 k/b F 1 nontransgenic and TLK transgenic mice. ( A ) Mice were immunized i.p. with 100 μg HEL in RIBI adjuvant. ( B ) Mice were immunized i.p. with 25 μg HEL-cGG in RIBI adjuvant. In both cases, day 35 data represent secondary responses 7 d after boosting with HEL/RIBI or HEL-cGG/ RIBI, respectively. HEL binding IgG was measured by ELISA in serum of individual mice ( dots ), and geometric means are shown in bars.

    Journal: The Journal of Experimental Medicine

    Article Title: Self-reactive B Cells Are Not Eliminated or Inactivated by Autoantigen Expressed on Thyroid Epithelial Cells

    doi:

    Figure Lengend Snippet: Anti-lysozyme IgG response in high-responder H-2 k/b F 1 nontransgenic and TLK transgenic mice. ( A ) Mice were immunized i.p. with 100 μg HEL in RIBI adjuvant. ( B ) Mice were immunized i.p. with 25 μg HEL-cGG in RIBI adjuvant. In both cases, day 35 data represent secondary responses 7 d after boosting with HEL/RIBI or HEL-cGG/ RIBI, respectively. HEL binding IgG was measured by ELISA in serum of individual mice ( dots ), and geometric means are shown in bars.

    Article Snippet: Bound anti-HEL IgG was developed with goat anti-mouse IgG (Fc specific) conjugated to alkaline phosphatase (catalog no. A-2429; Sigma), and bound anti-HEL IgMa was developed with RS3.1-biotin followed by streptavidin-alkaline phosphatase (Sigma).

    Techniques: Transgenic Assay, Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

    Antibody and T cell responses following vaccination with OVA targeted to MHC class II molecules or CCR1/3/5. (a,b) Supernatants of 293E cells transfected with the indicated plasmids were tested for secreted proteins in ELISA (a) and examined by Western blotting with anti-OVA mAb under reducing (+ME) or non-reducing (-ME) conditions (b). Vaccine proteins are indicated below lanes. (c-f) Mice were immunized once i.d. with 25 µg DNA/EP, as indicated. (c-e) Sera were assayed for total IgG (c), IgG1 (d) or IgG2a (e) against OVA. (f) Splenocytes collected at day 14 post immunization were stimulated in vitro with OVA protein or controls as indicated, and analyzed by an IFNγ EliSpot. *indicates p

    Journal: PLoS ONE

    Article Title: The Specificity of Targeted Vaccines for APC Surface Molecules Influences the Immune Response Phenotype

    doi: 10.1371/journal.pone.0080008

    Figure Lengend Snippet: Antibody and T cell responses following vaccination with OVA targeted to MHC class II molecules or CCR1/3/5. (a,b) Supernatants of 293E cells transfected with the indicated plasmids were tested for secreted proteins in ELISA (a) and examined by Western blotting with anti-OVA mAb under reducing (+ME) or non-reducing (-ME) conditions (b). Vaccine proteins are indicated below lanes. (c-f) Mice were immunized once i.d. with 25 µg DNA/EP, as indicated. (c-e) Sera were assayed for total IgG (c), IgG1 (d) or IgG2a (e) against OVA. (f) Splenocytes collected at day 14 post immunization were stimulated in vitro with OVA protein or controls as indicated, and analyzed by an IFNγ EliSpot. *indicates p

    Article Snippet: Detection of serum anti-HA or anti-OVA antibodies Sandwich ELISAs were performed with either inactivated A/PR/8/34 (H1N1) (PR8) virus (Charles River) (1∶1600 in PBS) or OVA protein (A5503, Sigma) (2 µg/ml) as coat, and detected with biotinylated anti-IgG (A2429, Sigma Aldrich), anti-IgG1a (553599, BD Pharmingen), anti-IgG2aa (553502, BD Pharmingen), anti-IgG2b (553393, BD Pharmingen) or anti-IgG3 (406803, BioLegend), as previously described .

    Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Mouse Assay, In Vitro, Enzyme-linked Immunospot

    TrkA and APP interaction in septal primary neurons measured by proximity ligation assay. (A) Confocal microscopy analysis of double-staining of APP (rabbit APP-CT A8717, red) and Trk (mouse Trk B3, green) in primary septal neurons showing co-localization of APP and TrkA. (B) The TrkA/APP complex was visualized by PLA, which generates red dots when the two proteins are in close proximity. Mouse and rabbit anti TrkA-CT (mouse Trk B3 and rabbit TrkA ab7261) and anti APP-CT (rabbit APP-CT A8717 and mouse APP clone C1-6.1) were used as primary antibodies and anti-mouse MINUS and anti-rabbit PLUS were used as secondary antibodies. Red fluorescent dot represents single interaction between TrkA and APP. (C) In addition to PLA (red dots), rat primary septal neurons were immunostained with goat anti ChAt (green) and with DAPI for nuclei (blue). (D) PLA assay using mouse anti APP (C1-6.1) and rabbit anti TrkB (sc-119) or TrkC (sc-117).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Association of TrkA and APP Is Promoted by NGF and Reduced by Cell Death-Promoting Agents

    doi: 10.3389/fnmol.2017.00015

    Figure Lengend Snippet: TrkA and APP interaction in septal primary neurons measured by proximity ligation assay. (A) Confocal microscopy analysis of double-staining of APP (rabbit APP-CT A8717, red) and Trk (mouse Trk B3, green) in primary septal neurons showing co-localization of APP and TrkA. (B) The TrkA/APP complex was visualized by PLA, which generates red dots when the two proteins are in close proximity. Mouse and rabbit anti TrkA-CT (mouse Trk B3 and rabbit TrkA ab7261) and anti APP-CT (rabbit APP-CT A8717 and mouse APP clone C1-6.1) were used as primary antibodies and anti-mouse MINUS and anti-rabbit PLUS were used as secondary antibodies. Red fluorescent dot represents single interaction between TrkA and APP. (C) In addition to PLA (red dots), rat primary septal neurons were immunostained with goat anti ChAt (green) and with DAPI for nuclei (blue). (D) PLA assay using mouse anti APP (C1-6.1) and rabbit anti TrkB (sc-119) or TrkC (sc-117).

    Article Snippet: Coverslips were washed three times for 5 min PBS 1X and then incubated with PLA Probe Anti-mouse MINUS (DUO92004) and Anti-rabbit PLUS (DUO92002) for 1 h at 37°C.

    Techniques: Proximity Ligation Assay, Confocal Microscopy, Double Staining

    SIM1 measures degranulation from natural killer (NK) and CD8 T cells . (A) Percent degranulating cells were measured using anti-CD107a antibodies SIM1, H4A3, and LS-C8580, with or without YAC-1 target cells as a stimulus. One representative experiment of four is shown (B) . Staining of the BWZ.CD107a-FLAG cell line with SIM1, H4A3, LS-C8580, or ID48 antibody clones. BWZ.36 cells transfected with irrelevant FLAG-tagged antigen (BWZ.NKR-P1G-FLAG) was included as a negative control, (C) enriched NK cells were stimulated with plate bound anti-NKp46, anti-NKR-P1A, or mouse IgG1 as isotype control, and SIM1 was used to measure percentage degranulating cells. NKp46 + (Wen23) or NKR-P1A bright (3.2.3) cells were gated on. (D) TCR + cells were sorted (purity 99%) and cultured in IL-2 for 2–3 days. The cells were then stimulated with plate bound anti-CD3 and anti-CD2 or mouse IgM isotype control for anti-CD3. The lower panel shows stimulation with anti-CD3 and anti-CD2 and staining with a SIM1 isotype control (FITC hamster IgG polyclonal). T cells were sorted using R73-Alexa647 and 3.2.3-PB antibodies, gating on R73 + /3.2.3 − cells. Data shown are from one representative experiment of three separate independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Degranulation Response in Cytotoxic Rat Lymphocytes Measured with a Novel CD107a Antibody

    doi: 10.3389/fimmu.2016.00572

    Figure Lengend Snippet: SIM1 measures degranulation from natural killer (NK) and CD8 T cells . (A) Percent degranulating cells were measured using anti-CD107a antibodies SIM1, H4A3, and LS-C8580, with or without YAC-1 target cells as a stimulus. One representative experiment of four is shown (B) . Staining of the BWZ.CD107a-FLAG cell line with SIM1, H4A3, LS-C8580, or ID48 antibody clones. BWZ.36 cells transfected with irrelevant FLAG-tagged antigen (BWZ.NKR-P1G-FLAG) was included as a negative control, (C) enriched NK cells were stimulated with plate bound anti-NKp46, anti-NKR-P1A, or mouse IgG1 as isotype control, and SIM1 was used to measure percentage degranulating cells. NKp46 + (Wen23) or NKR-P1A bright (3.2.3) cells were gated on. (D) TCR + cells were sorted (purity 99%) and cultured in IL-2 for 2–3 days. The cells were then stimulated with plate bound anti-CD3 and anti-CD2 or mouse IgM isotype control for anti-CD3. The lower panel shows stimulation with anti-CD3 and anti-CD2 and staining with a SIM1 isotype control (FITC hamster IgG polyclonal). T cells were sorted using R73-Alexa647 and 3.2.3-PB antibodies, gating on R73 + /3.2.3 − cells. Data shown are from one representative experiment of three separate independent experiments.

    Article Snippet: Anti-FLAG antibody (M2, unconjugated) and anti-mouse IgG (goat polyclonal-FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Staining, Clone Assay, Transfection, Negative Control, Cell Culture

    Expression of human leucocyte antigen (HLA)-B27 (a) and monomorphic major histocompatibility complex (MHC) class I (b) molecules on monocytes from seven patients with Salmonella -triggered reactive arthritis (ReA) who were positive for the HLA-B27 gene during the acute phase of infection (1–4 weeks, patient 3 after 8 weeks) and during the follow-up. *Patients ◊ and ▪ did not recover during the follow-up. The cells were incubated with monoclonal antibodies (mAbs) specific for HLA-B27 (HLA-ABC-m3) and monomorphic MHC class I (w6/32) and then with fluorescein isothiocyanate (FITC)-labelled F(ab′) 2 fragments of goat antimouse IgG. The y -axis is the mean fluorescence intensity (MFI) of the specific mAb. The MFI of the negative control mAb was subtracted from each value before plotting.

    Journal: Immunology

    Article Title: Enterobacterial infection modulates major histocompatibility complex class I expression on mononuclear cells

    doi: 10.1046/j.1365-2567.1999.00803.x

    Figure Lengend Snippet: Expression of human leucocyte antigen (HLA)-B27 (a) and monomorphic major histocompatibility complex (MHC) class I (b) molecules on monocytes from seven patients with Salmonella -triggered reactive arthritis (ReA) who were positive for the HLA-B27 gene during the acute phase of infection (1–4 weeks, patient 3 after 8 weeks) and during the follow-up. *Patients ◊ and ▪ did not recover during the follow-up. The cells were incubated with monoclonal antibodies (mAbs) specific for HLA-B27 (HLA-ABC-m3) and monomorphic MHC class I (w6/32) and then with fluorescein isothiocyanate (FITC)-labelled F(ab′) 2 fragments of goat antimouse IgG. The y -axis is the mean fluorescence intensity (MFI) of the specific mAb. The MFI of the negative control mAb was subtracted from each value before plotting.

    Article Snippet: Cells were then washed twice and incubated with fluorescein isothiocyanate (FITC)-labelled F(ab′)2 fragments of goat antimouse IgG (1:200 v/v) at +4° for 15 min (Sigma Chemical Company, St Louis, MO).

    Techniques: Expressing, Infection, Incubation, Fluorescence, Negative Control

    (a) Expression of human leucocyte antigen (HLA)-B27 and major histocompatibility complex (MHC) class I molecules on monocytes from the patient with uncomplicated Salmonella infection. The cells were incubated with monoclonal antibodies (mAbs) specific for HLA-B27 or with negative control mAbs, and then with fluorescein isothiocyanate (FITC)-labelled F(ab′) 2 fragments of goat antimouse IgG. The expression of HLA-B27 detected with mAb HLA-ABC-m3 is shown as black histograms and corresponding negative controls as white histograms. The x -axis is the mean fluorescence intensity (MFI) on a log scale and the y -axis is the relative number of the cells. (b) Expression of monomorphic MHC class I on monocytes from patients with S. enteritidis infection without reactive arthritis (ReA). The cells were incubated with mAb recognizing monomorphic MHC class I and then with FITC-labelled F(ab′) 2 fragments of goat antimouse IgG. The y -axis is the MFI of the specific mAb. The MFI of the negative control mAb was subtracted from each value before plotting. (c) Expression of HLA-ABC-m3 and FD705 epitopes of HLA-B27 on monocytes from a healthy control subject (A, B and C). The second sample (B) was taken 5 weeks and the third sample (C) 1 year after the first sample (A).

    Journal: Immunology

    Article Title: Enterobacterial infection modulates major histocompatibility complex class I expression on mononuclear cells

    doi: 10.1046/j.1365-2567.1999.00803.x

    Figure Lengend Snippet: (a) Expression of human leucocyte antigen (HLA)-B27 and major histocompatibility complex (MHC) class I molecules on monocytes from the patient with uncomplicated Salmonella infection. The cells were incubated with monoclonal antibodies (mAbs) specific for HLA-B27 or with negative control mAbs, and then with fluorescein isothiocyanate (FITC)-labelled F(ab′) 2 fragments of goat antimouse IgG. The expression of HLA-B27 detected with mAb HLA-ABC-m3 is shown as black histograms and corresponding negative controls as white histograms. The x -axis is the mean fluorescence intensity (MFI) on a log scale and the y -axis is the relative number of the cells. (b) Expression of monomorphic MHC class I on monocytes from patients with S. enteritidis infection without reactive arthritis (ReA). The cells were incubated with mAb recognizing monomorphic MHC class I and then with FITC-labelled F(ab′) 2 fragments of goat antimouse IgG. The y -axis is the MFI of the specific mAb. The MFI of the negative control mAb was subtracted from each value before plotting. (c) Expression of HLA-ABC-m3 and FD705 epitopes of HLA-B27 on monocytes from a healthy control subject (A, B and C). The second sample (B) was taken 5 weeks and the third sample (C) 1 year after the first sample (A).

    Article Snippet: Cells were then washed twice and incubated with fluorescein isothiocyanate (FITC)-labelled F(ab′)2 fragments of goat antimouse IgG (1:200 v/v) at +4° for 15 min (Sigma Chemical Company, St Louis, MO).

    Techniques: Expressing, Infection, Incubation, Negative Control, Fluorescence

    (a) Expression of human leucocyte antigen (HLA)-B27, HLA-A2, HLA-B7 and major histocompatibility complex (MHC) class I molecules on monocytes from patient 5 (P5) with Salmonella -triggered reactive arthritis (ReA) at the start of the infection and during the follow-up. The cells were incubated with monoclonal antibodies (mAbs) specific for HLA-B27 (three mAbs against different epitopes of HLA-B27), HLA-A2, HLA-B7 and monomorphic MHC class I and then with fluorescein isothiocyanate (FITC)-labelled F(ab′) 2 fragments of goat antimouse IgG. The expression of MHC class I epitopes recognized with mAbs is shown as black histograms and corresponding negative controls as white histograms. The x -axis is the mean fluorescence intensity (MFI) on a log scale, and the y -axis is the relative number of the cells. (b) Expression of HLA-B27 and monomorphic MHC class I epitopes on monocytes from a patient with Yersinia -triggered ReA. The cells were incubated with mAbs specific for HLA-B27 (four mAbs against different epitopes of HLA-B27) and monomorphic MHC class I and then with FITC-labelled F(ab′) 2 fragments of goat antimouse IgG.

    Journal: Immunology

    Article Title: Enterobacterial infection modulates major histocompatibility complex class I expression on mononuclear cells

    doi: 10.1046/j.1365-2567.1999.00803.x

    Figure Lengend Snippet: (a) Expression of human leucocyte antigen (HLA)-B27, HLA-A2, HLA-B7 and major histocompatibility complex (MHC) class I molecules on monocytes from patient 5 (P5) with Salmonella -triggered reactive arthritis (ReA) at the start of the infection and during the follow-up. The cells were incubated with monoclonal antibodies (mAbs) specific for HLA-B27 (three mAbs against different epitopes of HLA-B27), HLA-A2, HLA-B7 and monomorphic MHC class I and then with fluorescein isothiocyanate (FITC)-labelled F(ab′) 2 fragments of goat antimouse IgG. The expression of MHC class I epitopes recognized with mAbs is shown as black histograms and corresponding negative controls as white histograms. The x -axis is the mean fluorescence intensity (MFI) on a log scale, and the y -axis is the relative number of the cells. (b) Expression of HLA-B27 and monomorphic MHC class I epitopes on monocytes from a patient with Yersinia -triggered ReA. The cells were incubated with mAbs specific for HLA-B27 (four mAbs against different epitopes of HLA-B27) and monomorphic MHC class I and then with FITC-labelled F(ab′) 2 fragments of goat antimouse IgG.

    Article Snippet: Cells were then washed twice and incubated with fluorescein isothiocyanate (FITC)-labelled F(ab′)2 fragments of goat antimouse IgG (1:200 v/v) at +4° for 15 min (Sigma Chemical Company, St Louis, MO).

    Techniques: Expressing, Infection, Incubation, Fluorescence

    SDS-PAGE and Western blot analysis of purified recombinant NS1 protein expressed in E . coli . Lane M, molecular mass markers; lane 1, SDS-PAGE analysis of purified recombinant NS1 protein; Lane 2, the recombinant NS1 protein was analyzed by western blotting using SPF chicken serum for the negative control. Lane 3, western blotting of the purified recombinant NS1 protein, with chicken anti-AIV antibody and horseradish peroxidase (HRP)-labeled sheep anti-chicken IgG H+L as the first and second antibody, respectively. The arrowhead indicates the position of recombinant NS1 protein (approximate molecular mass of 30 kDa).

    Journal: PLoS ONE

    Article Title: Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

    doi: 10.1371/journal.pone.0149868

    Figure Lengend Snippet: SDS-PAGE and Western blot analysis of purified recombinant NS1 protein expressed in E . coli . Lane M, molecular mass markers; lane 1, SDS-PAGE analysis of purified recombinant NS1 protein; Lane 2, the recombinant NS1 protein was analyzed by western blotting using SPF chicken serum for the negative control. Lane 3, western blotting of the purified recombinant NS1 protein, with chicken anti-AIV antibody and horseradish peroxidase (HRP)-labeled sheep anti-chicken IgG H+L as the first and second antibody, respectively. The arrowhead indicates the position of recombinant NS1 protein (approximate molecular mass of 30 kDa).

    Article Snippet: After blocking with 5% nonfat milk in PBS overnight at 4°C, the membrane was incubated with MAb D7 (diluted 1:2,000 in PBS) at 37°C for 1 h, washed three times with PBS containing 0.05% (w/v) Tween 20 (PBST, pH 7.4), and probed with a 1:5,000 dilution of HRP-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO, USA) at 37°C for 1 h. Reactivity was visualized with the substrate 3, 3'-diaminobenzidine (DAB, Sigma).

    Techniques: SDS Page, Western Blot, Purification, Recombinant, Negative Control, Labeling

    Expression and immunolocalization of Ts -Pmy in the different developmental stages of T. spiralis . (A) RT-PCR showing mRNA transcription of Ts -Pmy and GAPDH (control) from the different developmental stages of T. spiralis . Lane 1, DNA Marker; Lane 2, ML; Lane 4, adult worm; Lane 6, NBL. Lanes 3, 5 and 7: non-reverse transcriptase negative controls. (B) Western blot detection of native Ts -Pmy and GAPDH (control) expressed in different developmental stages of T. spiralis by specific antibodies with the Odyssey two color infrared imaging system. Lane 1, ML extracts (8 µg); Lane 2, adult worm extracts (8 µg); Lane 3, NBL extracts (8 µg); Lane 4, purified r Ts -Pmy (500 ng). (C). Electron micrographs of T. spiralis immunolabeled with anti-r Ts- Pmy mAb. NBL (a) and adult worm (b) were incubated with mouse anti-r Ts- Pmy mAb or normal mouse serum (c and d) and then with gold-conjugated protein A. Scale bars represent 0.2 µm. (D) Immunofluorescence micrographs of intact T. spiralis immunolabeled with rabbit antiserum against Ts -Pmy. Intact whole adult worms (a) and NBL (b) were incubated with anti-r Ts- Pmy rabbit serum or normal rabbit serum (c and d) and then with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Scale bars represent 20 µm.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Trichinella spiralis Paramyosin Binds to C8 and C9 and Protects the Tissue-Dwelling Nematode from Being Attacked by Host Complement

    doi: 10.1371/journal.pntd.0001225

    Figure Lengend Snippet: Expression and immunolocalization of Ts -Pmy in the different developmental stages of T. spiralis . (A) RT-PCR showing mRNA transcription of Ts -Pmy and GAPDH (control) from the different developmental stages of T. spiralis . Lane 1, DNA Marker; Lane 2, ML; Lane 4, adult worm; Lane 6, NBL. Lanes 3, 5 and 7: non-reverse transcriptase negative controls. (B) Western blot detection of native Ts -Pmy and GAPDH (control) expressed in different developmental stages of T. spiralis by specific antibodies with the Odyssey two color infrared imaging system. Lane 1, ML extracts (8 µg); Lane 2, adult worm extracts (8 µg); Lane 3, NBL extracts (8 µg); Lane 4, purified r Ts -Pmy (500 ng). (C). Electron micrographs of T. spiralis immunolabeled with anti-r Ts- Pmy mAb. NBL (a) and adult worm (b) were incubated with mouse anti-r Ts- Pmy mAb or normal mouse serum (c and d) and then with gold-conjugated protein A. Scale bars represent 0.2 µm. (D) Immunofluorescence micrographs of intact T. spiralis immunolabeled with rabbit antiserum against Ts -Pmy. Intact whole adult worms (a) and NBL (b) were incubated with anti-r Ts- Pmy rabbit serum or normal rabbit serum (c and d) and then with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Scale bars represent 20 µm.

    Article Snippet: After being blocked with a 5% (w/v) skim milk solution in Tris-buffered saline (TBS) containing 0.05% Tween-20 (Sigma, USA) (TBS-T) for 1 hour at room temperature, the membrane was incubated with mAb 7E2 at a concentration of 50–100 ng/mL in TBS-T. Peroxidase-conjugated goat anti-mouse IgG (1∶5,000; Sigma, USA) was used as secondary antibody.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Western Blot, Imaging, Purification, Immunolabeling, Incubation, Immunofluorescence

    A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.

    Journal: The Journal of Neuroscience

    Article Title: Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells

    doi: 10.1523/JNEUROSCI.21-22-08915.2001

    Figure Lengend Snippet: A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.

    Article Snippet: Brefeldin A, cytochrome c , monensin, wortmannin, and secondary anti-chicken antibody (M8645) were from Sigma (St. Louis, MO), Ilford L4 emulsion was from Polysciences (Warrington, PA), K252a was from Alomone Labs, and the PI3-kinase inhibitor LY294,002 was from Alexis Corp (San Diego, CA).

    Techniques: Activity Assay, Injection, Blocking Assay, Derivative Assay

    Assembly is restricted between the members of the TREK/TRAAK subfamily. ( A ) Phylogenetic tree of human K 2P subunits. The tree was constructed with a multiple alignment using fast Fourier transform (MAFFT) program. ( B ) Duolink in situ PLA between TREK1 and other K 2P subunits representative of the different subfamilies. HA-TREK1 was coexpressed in MDCK cells with Myc-tagged subunits. Percentage of positive cells corresponds to the number of PLA-positive cells relative to the number of transfected cells. Number of independent experiments is given in parenthesis and corresponds to > 300 cells analyzed per condition. Data were analyzed by using Mann–Whitney test: * P

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

    Article Title: Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties

    doi: 10.1073/pnas.1522748113

    Figure Lengend Snippet: Assembly is restricted between the members of the TREK/TRAAK subfamily. ( A ) Phylogenetic tree of human K 2P subunits. The tree was constructed with a multiple alignment using fast Fourier transform (MAFFT) program. ( B ) Duolink in situ PLA between TREK1 and other K 2P subunits representative of the different subfamilies. HA-TREK1 was coexpressed in MDCK cells with Myc-tagged subunits. Percentage of positive cells corresponds to the number of PLA-positive cells relative to the number of transfected cells. Number of independent experiments is given in parenthesis and corresponds to > 300 cells analyzed per condition. Data were analyzed by using Mann–Whitney test: * P

    Article Snippet: PLA probe anti-Mouse PLUS (DUO92001), PLA probe anti-Rabbit MINUS (DUO92005), and Duolink In Situ Detection Reagent Red Kit (DUO92008) were purchased from Sigma-Aldrich.

    Techniques: Construct, In Situ, Proximity Ligation Assay, Transfection, MANN-WHITNEY

    Indirect PLA in cells. Cells co-expressing GPCR protomer A and GPCR protomer B are incubated with anti-mouse antibody selective for protomer A and anti-rabbit antibody selective for protomer B, followed by incubation with species-specific PLA probes PLUS and MINUS. Ligation of the oligonucleotide sequences creates a rolling circle template that is amplified by polymerase in the presence of fluorescent oligonucleotides and can be detected as red dots by confocal microscopy with the nuclei stained in blue by DAPI.

    Journal: Current protocols in pharmacology

    Article Title: Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay

    doi: 10.1002/cpph.15

    Figure Lengend Snippet: Indirect PLA in cells. Cells co-expressing GPCR protomer A and GPCR protomer B are incubated with anti-mouse antibody selective for protomer A and anti-rabbit antibody selective for protomer B, followed by incubation with species-specific PLA probes PLUS and MINUS. Ligation of the oligonucleotide sequences creates a rolling circle template that is amplified by polymerase in the presence of fluorescent oligonucleotides and can be detected as red dots by confocal microscopy with the nuclei stained in blue by DAPI.

    Article Snippet: Cells endogenously expressing one or both receptor protomers being investigated, plated and processed as in Support Protocol 2 PLA probes (species-specific secondary antibodies conjugated to either PLUS or MINUS oligonucleotide sequences): every PLA probe kit includes the blocking and antibody diluent solutions (Sigma-Aldrich, cat. no. DUO92001 to DUO 92010; check company Web site) in addition to the conjugated secondary antibodies (the method for determining the selectivity of the PLA probes is described in Critical Parameters) Primary antibodies from a commercial supplier or generated and validated in-house, that selectively recognize each protomer being investigated (described in Critical Parameters is a procedure for determining the selectivity of the antibodies for a target protomer) Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma-Aldrich, cat. no. DUO82049), or equivalent reagents prepared in-house (see recipes) 0.01× PLA Buffer B (see recipe) Duolink In Situ Detection Reagent Red (includes ligase, ligation reagent, polymerase, and amplification reagent; Sigma-Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site) Duolink In Situ Mounting medium with DAPI (Sigma-Aldrich, cat. no. DUO82040) Nail polish or acrylic resin Blotting paper Coverslips (glass) Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red) Additional reagents and equipment for plating and processing cells for PLA (Support Protocol 2) 1 Follow Support Protocol 2 for plating and processing cells for PLA.

    Techniques: Proximity Ligation Assay, Expressing, Incubation, Ligation, Amplification, Confocal Microscopy, Staining

    Schematic of single recognition PLA. In this modification of the PLA protocol, samples are incubated with an antibody that recognizes a single receptor protomer, followed by incubation with species specific PLA probes (i.e., a mixture of secondary antibodies coupled to oligonucleotides PLUS along with antibodies coupled to oligonucleotides MINUS).

    Journal: Current protocols in pharmacology

    Article Title: Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay

    doi: 10.1002/cpph.15

    Figure Lengend Snippet: Schematic of single recognition PLA. In this modification of the PLA protocol, samples are incubated with an antibody that recognizes a single receptor protomer, followed by incubation with species specific PLA probes (i.e., a mixture of secondary antibodies coupled to oligonucleotides PLUS along with antibodies coupled to oligonucleotides MINUS).

    Article Snippet: Cells endogenously expressing one or both receptor protomers being investigated, plated and processed as in Support Protocol 2 PLA probes (species-specific secondary antibodies conjugated to either PLUS or MINUS oligonucleotide sequences): every PLA probe kit includes the blocking and antibody diluent solutions (Sigma-Aldrich, cat. no. DUO92001 to DUO 92010; check company Web site) in addition to the conjugated secondary antibodies (the method for determining the selectivity of the PLA probes is described in Critical Parameters) Primary antibodies from a commercial supplier or generated and validated in-house, that selectively recognize each protomer being investigated (described in Critical Parameters is a procedure for determining the selectivity of the antibodies for a target protomer) Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma-Aldrich, cat. no. DUO82049), or equivalent reagents prepared in-house (see recipes) 0.01× PLA Buffer B (see recipe) Duolink In Situ Detection Reagent Red (includes ligase, ligation reagent, polymerase, and amplification reagent; Sigma-Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site) Duolink In Situ Mounting medium with DAPI (Sigma-Aldrich, cat. no. DUO82040) Nail polish or acrylic resin Blotting paper Coverslips (glass) Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red) Additional reagents and equipment for plating and processing cells for PLA (Support Protocol 2) 1 Follow Support Protocol 2 for plating and processing cells for PLA.

    Techniques: Proximity Ligation Assay, Modification, Incubation

    Schematic of direct PLA. Antibodies that selectively recognize one receptor protomer are conjugated directly with the PLA oligonucleotide PLUS while the antibodies that recognize the other receptor protomer are conjugated directly with the PLA oligonucleotide MINUS using commercially available probemakers. The PLA assay is then carried out in the absence of secondary antibodies.

    Journal: Current protocols in pharmacology

    Article Title: Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay

    doi: 10.1002/cpph.15

    Figure Lengend Snippet: Schematic of direct PLA. Antibodies that selectively recognize one receptor protomer are conjugated directly with the PLA oligonucleotide PLUS while the antibodies that recognize the other receptor protomer are conjugated directly with the PLA oligonucleotide MINUS using commercially available probemakers. The PLA assay is then carried out in the absence of secondary antibodies.

    Article Snippet: Cells endogenously expressing one or both receptor protomers being investigated, plated and processed as in Support Protocol 2 PLA probes (species-specific secondary antibodies conjugated to either PLUS or MINUS oligonucleotide sequences): every PLA probe kit includes the blocking and antibody diluent solutions (Sigma-Aldrich, cat. no. DUO92001 to DUO 92010; check company Web site) in addition to the conjugated secondary antibodies (the method for determining the selectivity of the PLA probes is described in Critical Parameters) Primary antibodies from a commercial supplier or generated and validated in-house, that selectively recognize each protomer being investigated (described in Critical Parameters is a procedure for determining the selectivity of the antibodies for a target protomer) Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma-Aldrich, cat. no. DUO82049), or equivalent reagents prepared in-house (see recipes) 0.01× PLA Buffer B (see recipe) Duolink In Situ Detection Reagent Red (includes ligase, ligation reagent, polymerase, and amplification reagent; Sigma-Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site) Duolink In Situ Mounting medium with DAPI (Sigma-Aldrich, cat. no. DUO82040) Nail polish or acrylic resin Blotting paper Coverslips (glass) Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red) Additional reagents and equipment for plating and processing cells for PLA (Support Protocol 2) 1 Follow Support Protocol 2 for plating and processing cells for PLA.

    Techniques: Proximity Ligation Assay

    Indirect PLA in a mouse brain slice. Mouse brain slices are incubated with anti-goat antibody selective for protomer A and anti-rabbit antibody selective for protomer B, followed by incubation with species-specific PLA probes PLUS and MINUS. Ligation of the oligonucleotide sequences creates a rolling circle template that is amplified by polymerase in the presence of fluorescent oligonucleotides and that can be detected as red dots by confocal microscopy. (Left Panel) Positive PLA signal (red dots) with the nuclei stained in blue by DAPI. (Upper Right Panel) Higher magnification of a cell devoid of PLA signal. (Lower Right Panel) Higher magnification of a cell positive for PLA signal.

    Journal: Current protocols in pharmacology

    Article Title: Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay

    doi: 10.1002/cpph.15

    Figure Lengend Snippet: Indirect PLA in a mouse brain slice. Mouse brain slices are incubated with anti-goat antibody selective for protomer A and anti-rabbit antibody selective for protomer B, followed by incubation with species-specific PLA probes PLUS and MINUS. Ligation of the oligonucleotide sequences creates a rolling circle template that is amplified by polymerase in the presence of fluorescent oligonucleotides and that can be detected as red dots by confocal microscopy. (Left Panel) Positive PLA signal (red dots) with the nuclei stained in blue by DAPI. (Upper Right Panel) Higher magnification of a cell devoid of PLA signal. (Lower Right Panel) Higher magnification of a cell positive for PLA signal.

    Article Snippet: Cells endogenously expressing one or both receptor protomers being investigated, plated and processed as in Support Protocol 2 PLA probes (species-specific secondary antibodies conjugated to either PLUS or MINUS oligonucleotide sequences): every PLA probe kit includes the blocking and antibody diluent solutions (Sigma-Aldrich, cat. no. DUO92001 to DUO 92010; check company Web site) in addition to the conjugated secondary antibodies (the method for determining the selectivity of the PLA probes is described in Critical Parameters) Primary antibodies from a commercial supplier or generated and validated in-house, that selectively recognize each protomer being investigated (described in Critical Parameters is a procedure for determining the selectivity of the antibodies for a target protomer) Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma-Aldrich, cat. no. DUO82049), or equivalent reagents prepared in-house (see recipes) 0.01× PLA Buffer B (see recipe) Duolink In Situ Detection Reagent Red (includes ligase, ligation reagent, polymerase, and amplification reagent; Sigma-Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site) Duolink In Situ Mounting medium with DAPI (Sigma-Aldrich, cat. no. DUO82040) Nail polish or acrylic resin Blotting paper Coverslips (glass) Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red) Additional reagents and equipment for plating and processing cells for PLA (Support Protocol 2) 1 Follow Support Protocol 2 for plating and processing cells for PLA.

    Techniques: Proximity Ligation Assay, Slice Preparation, Incubation, Ligation, Amplification, Confocal Microscopy, Staining

    Schematic representation of indirect PLA to detect receptor heteromers. Samples are first incubated with antibodies that selectively recognize each receptor protomer. This is followed by incubation with species-specific secondary antibodies coupled to oligonucleotide sequences (i.e., the PLA probes PLUS and MINUS). Ligation of the oligonucleotide sequences creates a rolling circle template that is amplified by polymerase in the presence of fluorescent oligonucleotides.

    Journal: Current protocols in pharmacology

    Article Title: Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay

    doi: 10.1002/cpph.15

    Figure Lengend Snippet: Schematic representation of indirect PLA to detect receptor heteromers. Samples are first incubated with antibodies that selectively recognize each receptor protomer. This is followed by incubation with species-specific secondary antibodies coupled to oligonucleotide sequences (i.e., the PLA probes PLUS and MINUS). Ligation of the oligonucleotide sequences creates a rolling circle template that is amplified by polymerase in the presence of fluorescent oligonucleotides.

    Article Snippet: Cells endogenously expressing one or both receptor protomers being investigated, plated and processed as in Support Protocol 2 PLA probes (species-specific secondary antibodies conjugated to either PLUS or MINUS oligonucleotide sequences): every PLA probe kit includes the blocking and antibody diluent solutions (Sigma-Aldrich, cat. no. DUO92001 to DUO 92010; check company Web site) in addition to the conjugated secondary antibodies (the method for determining the selectivity of the PLA probes is described in Critical Parameters) Primary antibodies from a commercial supplier or generated and validated in-house, that selectively recognize each protomer being investigated (described in Critical Parameters is a procedure for determining the selectivity of the antibodies for a target protomer) Duolink In Situ Wash Buffer (Fluorescence) comprising PLA Buffer A and Buffer B (Sigma-Aldrich, cat. no. DUO82049), or equivalent reagents prepared in-house (see recipes) 0.01× PLA Buffer B (see recipe) Duolink In Situ Detection Reagent Red (includes ligase, ligation reagent, polymerase, and amplification reagent; Sigma-Aldrich, cat. no. DUO92008; for fluorophores different from Red, check the Web site) Duolink In Situ Mounting medium with DAPI (Sigma-Aldrich, cat. no. DUO82040) Nail polish or acrylic resin Blotting paper Coverslips (glass) Leica TCS SP5 confocal laser scanning microscope (405 nm laser line for DAPI, 568 nm laser line for Detection Reagent Red) Additional reagents and equipment for plating and processing cells for PLA (Support Protocol 2) 1 Follow Support Protocol 2 for plating and processing cells for PLA.

    Techniques: Proximity Ligation Assay, Incubation, Ligation, Amplification