goat anti mouse igg conjugated to alexa fluor 488  (Thermo Fisher)


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    Goat anti Mouse IgG
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    PA1-32125
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    Structured Review

    Thermo Fisher goat anti mouse igg conjugated to alexa fluor 488
    Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an <t>Alexa</t> Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p

    https://www.bioz.com/result/goat anti mouse igg conjugated to alexa fluor 488/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti mouse igg conjugated to alexa fluor 488 - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells"

    Article Title: Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

    Journal: Stem Cells International

    doi: 10.1155/2018/1203717

    Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p
    Figure Legend Snippet: Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p

    Techniques Used: Expressing, Confocal Microscopy, Transfection, Staining, Western Blot

    2) Product Images from "Comparative Assessment of Transmission-Blocking Vaccine Candidates against Plasmodium falciparum"

    Article Title: Comparative Assessment of Transmission-Blocking Vaccine Candidates against Plasmodium falciparum

    Journal: Scientific Reports

    doi: 10.1038/srep11193

    Reactivity of vaccine-induced antibodies against Pfs230-C, Pfs25 and Pfs48/45+ NGln to native parasite antigen. In vitro cultured Pfs25DR3 transgenic P. berghei ookinetes or P. falciparum NF54 microgametes and macrogametes were stained with purified total IgG from pooled serum ( n = 5) against Pfs25 (top panel) or antisera against Pfs230-C (middle panel) and Pfs48/45 +NGln (bottom panel). Antibody binding was detected by Alexa Fluor® 488-conjugated goat anti-mouse IgG (green) and the DNA was stained with DAPI (blue). Scale bars, 2 μm.
    Figure Legend Snippet: Reactivity of vaccine-induced antibodies against Pfs230-C, Pfs25 and Pfs48/45+ NGln to native parasite antigen. In vitro cultured Pfs25DR3 transgenic P. berghei ookinetes or P. falciparum NF54 microgametes and macrogametes were stained with purified total IgG from pooled serum ( n = 5) against Pfs25 (top panel) or antisera against Pfs230-C (middle panel) and Pfs48/45 +NGln (bottom panel). Antibody binding was detected by Alexa Fluor® 488-conjugated goat anti-mouse IgG (green) and the DNA was stained with DAPI (blue). Scale bars, 2 μm.

    Techniques Used: In Vitro, Cell Culture, Transgenic Assay, Staining, Purification, Binding Assay

    3) Product Images from "Microglia P2Y13 Receptors Prevent Astrocyte Proliferation Mediated by P2Y1 Receptors"

    Article Title: Microglia P2Y13 Receptors Prevent Astrocyte Proliferation Mediated by P2Y1 Receptors

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00418

    BrdU staining and its cellular localization in astrocyte cultures. Astrocyte cultures were incubated with solvent (A,D) , ADPβS (B,E) or 10% FBS, a positive control for cell proliferation (C,F) , for 48 h. BrdU (100 μM) was added to the medium for the last 24 h and then stained with rabbit anti-BrdU (Alexa Fluor 594, red) to visualize BrdU incorporation. Microglia were then co-labeled with mouse anti-Cd11b ( A–C ; Alexa Fluor 488, green) and astrocytes, with mouse anti-GFAP ( D–F ; Alexa Fluor 488, green). BrdU positive nuclei co-localize mainly with astrocytes, showing they are the main proliferating cells within the astrocyte cultures. Representative images from 3 different cultures. Scale bar: 100 μm.
    Figure Legend Snippet: BrdU staining and its cellular localization in astrocyte cultures. Astrocyte cultures were incubated with solvent (A,D) , ADPβS (B,E) or 10% FBS, a positive control for cell proliferation (C,F) , for 48 h. BrdU (100 μM) was added to the medium for the last 24 h and then stained with rabbit anti-BrdU (Alexa Fluor 594, red) to visualize BrdU incorporation. Microglia were then co-labeled with mouse anti-Cd11b ( A–C ; Alexa Fluor 488, green) and astrocytes, with mouse anti-GFAP ( D–F ; Alexa Fluor 488, green). BrdU positive nuclei co-localize mainly with astrocytes, showing they are the main proliferating cells within the astrocyte cultures. Representative images from 3 different cultures. Scale bar: 100 μm.

    Techniques Used: BrdU Staining, Incubation, Positive Control, Staining, BrdU Incorporation Assay, Labeling

    4) Product Images from "Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast *"

    Article Title: Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M710470200

    Pma1p is mislocalized to the vacuole in vma mutants lacking vacuolar protease activity. Pma1p was localized by immunofluorescence microscopy in both wild-type and vma mutants that contained a pep4-3 mutation, which inhibits most vacuolar protease activity. Nomarski images ( left panels ) and indirect immunofluorescence using anti-Pma1p antibody followed by Alexa Fluor 488-conjugated secondary antibody ( right panels ) are shown for each field. All fluorescence images were recorded with identical exposure times.
    Figure Legend Snippet: Pma1p is mislocalized to the vacuole in vma mutants lacking vacuolar protease activity. Pma1p was localized by immunofluorescence microscopy in both wild-type and vma mutants that contained a pep4-3 mutation, which inhibits most vacuolar protease activity. Nomarski images ( left panels ) and indirect immunofluorescence using anti-Pma1p antibody followed by Alexa Fluor 488-conjugated secondary antibody ( right panels ) are shown for each field. All fluorescence images were recorded with identical exposure times.

    Techniques Used: Activity Assay, Immunofluorescence, Microscopy, Mutagenesis, Fluorescence

    5) Product Images from "Antagonism of RNase L Is Required for Murine Coronavirus Replication in Kupffer Cells and Liver Sinusoidal Endothelial Cells but Not in Hepatocytes"

    Article Title: Antagonism of RNase L Is Required for Murine Coronavirus Replication in Kupffer Cells and Liver Sinusoidal Endothelial Cells but Not in Hepatocytes

    Journal: Journal of Virology

    doi: 10.1128/JVI.01423-16

    Isolation and characterization of hepatocytes (Hep), KC, and LSEC. Hep, KC, and LSEC were isolated as described in Materials and Methods. (A) Hep were stained with mouse anti-human HNF-4α followed by secondary Alexa Fluor 488-conjugated goat anti-mouse
    Figure Legend Snippet: Isolation and characterization of hepatocytes (Hep), KC, and LSEC. Hep, KC, and LSEC were isolated as described in Materials and Methods. (A) Hep were stained with mouse anti-human HNF-4α followed by secondary Alexa Fluor 488-conjugated goat anti-mouse

    Techniques Used: Isolation, Staining

    6) Product Images from "Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells"

    Article Title: Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

    Journal: Stem Cells International

    doi: 10.1155/2018/1203717

    Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p
    Figure Legend Snippet: Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p

    Techniques Used: Expressing, Confocal Microscopy, Transfection, Staining, Western Blot

    Related Articles

    Staining:

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

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

    Incubation:

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

    Article Title: Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1
    Article Snippet: .. Then, samples were incubated with anti-MBNL1 3A4 antibody (Santa Cruz Biotechnology) solution in PBS at room temperature for 1 h. After that samples were washed 3 times with PBS and incubated using goat anti-mouse IgG-Alexa Fluor 488 (Invitrogen, Thermo Scientific, Carlsbad, CA). .. After that, samples were incubated with Hoechst 33,258 (Invitrogen) in PBS at room temperature for 10 min.

    Labeling:

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

    other:

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

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    Thermo Fisher secondary antibody goat anti mouse igg alexa fluor 488 conjugate
    Pneumococcal lipoproteins are highly abundant on the pneumococcal surface. (A) In immunoblots, the specificity of antisera derived from intraperitoneal immunisations of CD-1 mice ( n = 6) with recombinant lipoproteins was assessed. Therefore, the wild-type strain S. pneumoniae D39Δ cps and the corresponding isogenic lipoprotein deficient mutants (2 × 10 8 bacteria per lane) were used. Enolase was detected with a rabbit anti-enolase serum and served as a loading control. (B) <t>IgG</t> antibody titrations were performed by incubating equimolar amounts of recombinant proteins with serial dilutions of isolated polyclonal IgGs. Detection was carried out using a peroxidase-coupled goat anti-mouse IgG followed by incubation with OPD as a substrate and absorbance was measured at 492 nm. Titrations were performed at least three times and the error bars represent the SEM. (C,D) Using the equation for the hyperbolic regression curve ( y [ A b s ] = B m a x · x K d + x , Bmax, maximal binding; Kd, concentration for half maximal binding) an initial IgG concentration was calculated in the linear dynamic range. The polyclonal IgGs with equal contents of IgG specific for each lipoprotein were therefore applied to enable the comparison of their surface abundances. In a flow cytometric approach, D39Δ cps (C) and D39 (D) were incubated with the appropriate calculated concentration of IgG and concentrations 5-, 10-, 20-, and 50-fold greater to analyse the surface abundance of the selected lipoproteins. Antibody binding was detected using a goat anti-mouse <t>Alexa</t> Fluor® 488-coupled secondary antibody. The percentage of positive gated events is depicted in the graphs, thereby indicating the proportion of wild-type bacteria positive for the binding of the respective anti-lipoprotein IgGs. The mean values of at least three independent experiments are shown, with error bars corresponding to SEM.
    Secondary Antibody Goat Anti Mouse Igg Alexa Fluor 488 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/secondary antibody goat anti mouse igg alexa fluor 488 conjugate/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
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    Thermo Fisher alexa fluor 488 conjugated goat anti rabbit igg
    Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5 ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to <t>Alexa</t> Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an <t>IgG</t> isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5 ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. ** P
    Alexa Fluor 488 Conjugated Goat Anti Rabbit Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 conjugated goat anti rabbit igg/product/Thermo Fisher
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    Thermo Fisher alexa fluor 488 conjugated goat anti mouse igg
    Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of <t>Alexa</t> Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous <t>IgG</t> ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p
    Alexa Fluor 488 Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 conjugated goat anti mouse igg/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
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    Thermo Fisher goat anti mouse igg conjugated to alexa fluor 488
    Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an <t>Alexa</t> Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p
    Goat Anti Mouse Igg Conjugated To Alexa Fluor 488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti mouse igg conjugated to alexa fluor 488/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Pneumococcal lipoproteins are highly abundant on the pneumococcal surface. (A) In immunoblots, the specificity of antisera derived from intraperitoneal immunisations of CD-1 mice ( n = 6) with recombinant lipoproteins was assessed. Therefore, the wild-type strain S. pneumoniae D39Δ cps and the corresponding isogenic lipoprotein deficient mutants (2 × 10 8 bacteria per lane) were used. Enolase was detected with a rabbit anti-enolase serum and served as a loading control. (B) IgG antibody titrations were performed by incubating equimolar amounts of recombinant proteins with serial dilutions of isolated polyclonal IgGs. Detection was carried out using a peroxidase-coupled goat anti-mouse IgG followed by incubation with OPD as a substrate and absorbance was measured at 492 nm. Titrations were performed at least three times and the error bars represent the SEM. (C,D) Using the equation for the hyperbolic regression curve ( y [ A b s ] = B m a x · x K d + x , Bmax, maximal binding; Kd, concentration for half maximal binding) an initial IgG concentration was calculated in the linear dynamic range. The polyclonal IgGs with equal contents of IgG specific for each lipoprotein were therefore applied to enable the comparison of their surface abundances. In a flow cytometric approach, D39Δ cps (C) and D39 (D) were incubated with the appropriate calculated concentration of IgG and concentrations 5-, 10-, 20-, and 50-fold greater to analyse the surface abundance of the selected lipoproteins. Antibody binding was detected using a goat anti-mouse Alexa Fluor® 488-coupled secondary antibody. The percentage of positive gated events is depicted in the graphs, thereby indicating the proportion of wild-type bacteria positive for the binding of the respective anti-lipoprotein IgGs. The mean values of at least three independent experiments are shown, with error bars corresponding to SEM.

    Journal: Frontiers in Immunology

    Article Title: Intranasal Vaccination With Lipoproteins Confers Protection Against Pneumococcal Colonisation

    doi: 10.3389/fimmu.2018.02405

    Figure Lengend Snippet: Pneumococcal lipoproteins are highly abundant on the pneumococcal surface. (A) In immunoblots, the specificity of antisera derived from intraperitoneal immunisations of CD-1 mice ( n = 6) with recombinant lipoproteins was assessed. Therefore, the wild-type strain S. pneumoniae D39Δ cps and the corresponding isogenic lipoprotein deficient mutants (2 × 10 8 bacteria per lane) were used. Enolase was detected with a rabbit anti-enolase serum and served as a loading control. (B) IgG antibody titrations were performed by incubating equimolar amounts of recombinant proteins with serial dilutions of isolated polyclonal IgGs. Detection was carried out using a peroxidase-coupled goat anti-mouse IgG followed by incubation with OPD as a substrate and absorbance was measured at 492 nm. Titrations were performed at least three times and the error bars represent the SEM. (C,D) Using the equation for the hyperbolic regression curve ( y [ A b s ] = B m a x · x K d + x , Bmax, maximal binding; Kd, concentration for half maximal binding) an initial IgG concentration was calculated in the linear dynamic range. The polyclonal IgGs with equal contents of IgG specific for each lipoprotein were therefore applied to enable the comparison of their surface abundances. In a flow cytometric approach, D39Δ cps (C) and D39 (D) were incubated with the appropriate calculated concentration of IgG and concentrations 5-, 10-, 20-, and 50-fold greater to analyse the surface abundance of the selected lipoproteins. Antibody binding was detected using a goat anti-mouse Alexa Fluor® 488-coupled secondary antibody. The percentage of positive gated events is depicted in the graphs, thereby indicating the proportion of wild-type bacteria positive for the binding of the respective anti-lipoprotein IgGs. The mean values of at least three independent experiments are shown, with error bars corresponding to SEM.

    Article Snippet: The bacteria were washed with PBS and stained using secondary antibody goat anti-mouse IgG Alexa-Fluor-488 conjugate (1:1,000; Thermo Fisher Scientific).

    Techniques: Western Blot, Derivative Assay, Mouse Assay, Recombinant, Isolation, Incubation, Binding Assay, Concentration Assay, Flow Cytometry

    Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5 ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5 ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. ** P

    Journal: Skeletal Muscle

    Article Title: Increased sphingosine-1-phosphate improves muscle regeneration in acutely injured mdx mice

    doi: 10.1186/2044-5040-3-20

    Figure Lengend Snippet: Direct injection results in elevated S1P levels which correlate with the activation of receptor 1 in muscle fibers. ( A ) To quantify the elevation of S1P following direct administration, we injected a single dose (same dose as Figure 5 ) of S1P in left TAs and vehicle in right TAs of uninjured mdx 4cv (n = 3, 11-MO) mice. TA muscles were harvested 15 minutes post injection for analysis by LC-MS/MS. Results indicate a significant elevation of S1P following direct injection. ( B ) To visualize the location of S1P following injection, biotinylated-S1P was injected in left TAs versus vehicle in right TAs of uninjured mdx 4cv mice (n = 2, 11-MO). Once more, TAs were harvested 15 minutes following injection. Staining with streptavidin conjugated to Alexa Fluor 594 reveals the presence of S1P-biotin around the perimeter of muscle fibers. ( C ) Staining of mdx 4cv TAs for S1PR1 and S1PR3 reveals S1PR1 is localized to the perimeter and perinuclear area (arrow) of muscle fibers (left photo). In contrast, staining for S1PR3 was mainly localized to the muscle vasculature (middle photo). Staining in parallel with an IgG isotype control for both antibodies shows the absence of non-specific staining (right graph). ( D ) Staining for S1PR1 in CTX-injured TAs (same tissue from Figure 5 ) reveals S1PR1 is present at the perimeter and perinuclear area of regenerating eMyHC+ fibers. ( E ) Staining for phosphorylated S1PR1 in the same mdx 4cv TAs was more prominent in the perinuclear area of eMyHC+ fibers, indicating the presence of active S1PR1 signaling in regenerating fibers. Scale bars = 50 μm. ** P

    Article Snippet: Following washes, Alexa Fluor 488 conjugated goat anti-rabbit IgG (Life Technologies) was administered at 1:800 dilution for 1 hour.

    Techniques: Injection, Activation Assay, Mouse Assay, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Staining

    Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p

    Journal: The Journal of Biological Chemistry

    Article Title: Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System *

    doi: 10.1074/jbc.M114.587253

    Figure Lengend Snippet: Treatment with iloprost prevents LPC-mediated vascular barrier disruption in the adult spinal cord. A , visualization of vascular leakage in the CNS. Upper panels, representative images of the thoracic spinal cord showing leakage of Alexa Fluor 555-conjugated cadaverine ( red ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. The broken line represents the outline of the tissue. Scale bar, 1 mm. Middle panels, representative images of the thoracic spinal cord showing the distribution of Alexa Fluor 555-conjugated cadaverine ( red ) in the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were counterstained for CD31 (vascular endothelial cell marker, green ). Scale bar, 100 μm. Lower panels, representative images of cross-sections of the thoracic spinal cord showing leakage of endogenous IgG ( green ) into the spinal cord 1 day after LPC injection. Iloprost treatment was initiated immediately after LPC injection. Sections were vascular counterstained with DyLight 594-labeled L. esculentum lectin (vascular endothelial cell marker, red ). Scale bar, 100 μm. B , quantification of Evans Blue leakage in lesions of the spinal cord at 1 day after the operation. Values represent the mean ± S.E. of three independent experiments. **, p

    Article Snippet: Sections were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500, Invitrogen, catalog no. A-11001) and DyLight 594-labeled L. esculentum (tomato) lectin (1:100) for 1 h at room temperature.

    Techniques: Injection, Marker, Labeling

    Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p

    Journal: Stem Cells International

    Article Title: Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells

    doi: 10.1155/2018/1203717

    Figure Lengend Snippet: Effect of Cx43 siRNA on CFTR protein localisation, expression, and function. (a) Confocal microscopy of cocultures after 6 days from the transfection with either siRNA against CX43 or scrambled siRNA. Cocultures were stained with an anti-CFTR primary antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green) and DAPI (blue). XZ images are shown. Original magnification: 60x. Scale bar: 10 μ m. (b) Representative Western blot of a typical experiment analysed using anti-human CFTR C-terminus antibody (dilution 1 : 500), anti-Cx43 (dilution 1 : 500), anti β -tubulin (dilution 1 : 1000). β -Tubulin was used to normalise protein loading. The histogram summarises the relative change in the ratio between band C and band B in the various conditions compared to the CFTR ratio in the 16HBE set as 1. Results represent means ± SEM of three experiments. ∗∗∗ p

    Article Snippet: Cells were incubated with an anti-CFTR (CF3) mouse monoclonal antibody (dilution 1 : 500; Abcam) followed by a goat anti-mouse IgG conjugated to Alexa Fluor 488 (dilution 1 : 1000; Invitrogen) used as secondary antibody or with an anti-Cx43 (CX-1B1) mouse monoclonal antibody and Alexa Fluor 488 conjugated (dilution 1 : 300; Abcam) in 0.2% BSA/PBS for 1 h on ice.

    Techniques: Expressing, Confocal Microscopy, Transfection, Staining, Western Blot