goat anti rabbit igg h l cross adsorbed secondary antibody  (Thermo Fisher)


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    Name:
    Goat anti Rabbit IgG H L Cross Adsorbed Secondary Antibody
    Description:
    Goat anti Rabbit IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    Catalog Number:
    31212
    Price:
    None
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher goat anti rabbit igg h l cross adsorbed secondary antibody
    Goat anti Rabbit IgG H L Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    https://www.bioz.com/result/goat anti rabbit igg h l cross adsorbed secondary antibody/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit igg h l cross adsorbed secondary antibody - by Bioz Stars, 2021-04
    97/100 stars

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    Related Articles

    Staining:

    Article Title: Photoactivatable Cre recombinase 3.0 for in vivo mouse applications
    Article Snippet: The whole body of mice were perfused and fixed wit 4% paraformaldehyde for 1 h immediately after end of restraint, and then the brains were harvested for cryosection. .. Amygdala sections were stained with primary antibodies for c-fos (polyclonal anti-rabbit, Synaptic Systems, Cat#226003) and mCherry (monoclonal anti-rat, 1:500, Invitrogen Cat16D7) and secondary antibodies (goat anti-rabbit Alexa 488 Cat#A11008, goat anti-rat Alexa 568, Invitrogen Cat#A11077) as well as nuclear stain Draq5 (5 µM, Thermo-Fisher Cat#62254). ..

    Incubation:

    Article Title: Myeloid Differentiation Primary Response 88–Cyclin D1 Signaling in Breast Cancer Cells Regulates Toll-Like Receptor 3-Mediated Cell Proliferation
    Article Snippet: Briefly, 40 × 104 cells were seeded on a coverslip in a 35-mm culture dish in complete media. .. Four hours before the addition of the TLR3 ligand, the MyD88 inhibitor was added and incubated for 24 h. For TLR3 surface expression, cells were fixed and allowed to bind with TLR 3 antibody (Invitrogen-PA5-29619) and Alexa 594-conjugated secondary anti-rabbit goat antibody (Invitrogen-A11012). .. For IL-6 expression, cells were fixed, permeabilized, and incubated with primary IL-6 antibody (Invitrogen-AMC0862) and Alexa 488-conjugated anti-mouse goat antibody (Invitrogen-A11001) and mounted with Vecta Shield-DAPI to counterstain the nuclei and were observed under fluorescence microscope (Leica DMI 6000B).

    Article Title: Docking of acetyl-CoA carboxylase to the plastid envelope membrane attenuates fatty acid production in plants
    Article Snippet: After three washes with PBS-GM, nonspecific binding was blocked with 1% bovine serum albumin in PBS-GM for 1 h. Protoplasts were then incubated overnight at 4 °C with rabbit anti-E37 antibodies (1:500) followed by 1 h incubation at room temperature with rat anti-HA antibodies (1:2000; Roche). .. Cells were washed for 20 min in PBS-GM, and incubated with goat anti-rabbit immunoglobulin G-Alexa Fluor 568 (1:1000; Molecular Probes) and with goat anti-rat immunoglobulin G-Alexa Fluor 488 (1:500; Molecular Probes) or with goat anti-mouse immunoglobulin G-Alexa Fluor 488 (1:500; Molecular Probes) for 1 h. Finally, cells were washed for 20 min in PBS and mounted using Vestashield mounting solution (Vector Laboratories). .. Cells were visualized with an inverted spectral confocal laser microscope (LEICA SP2-AOBS).

    Article Title: Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic colon lesions
    Article Snippet: .. Samples were then washed with PBS and incubated in secondary goat anti-rabbit Alexa-568 antibody (1:200, Invitrogen) and goat anti-mouse Alexa 488 antibody (1:200, Invitrogen) for 1 hour at room temperature (RT). .. Slides were washed in PBS and incubated with DAPI (2 min, RT; 1:10,000) for nuclear visualization.

    Article Title: Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence
    Article Snippet: A solution of 1% of Bovine Serum Albumin (BSA) (BP671-1, Fisher Scientific, USA) and 0.1% of Tween-20 (BP337-500, Fisher Scientific, USA) in PBS was prepared. .. Sample 2 was incubated for one hour in 5 μl of Alexa Fluor 532 goat anti-rabbit IgG (A-11009, Life Technologies, USA) diluted with 1 ml of the BSA, Tween-20, and PBS solution. .. BSA prevents nonspecific binding and Tween-20 enhances the permeability of the tissue.

    Article Title: Induction of Peroxisomes by Butyrate-Producing Probiotics
    Article Snippet: The sections were again washed twice with PBS and then incubated with blocking buffer (2% bovine serum albumin, 0.5% Triton X-100 in PBS) for 60 min, followed by incubation with rabbit antibody against peroxisome membrane protein 70 (anti-PMP70: S34201, 1:1000, Invitrogen; Carlsbad, CA, USA) overnight at 4°C. .. Sections were subsequently washed five times with PBS containing 0.5% Triton X-100 and incubated with Texas Alexa Fluor 488-conjugated goat anti-rabbit secondary antibodies (A-11008, 1:1000, Invitrogen) for 60 min at room temperature. ..

    Expressing:

    Article Title: Myeloid Differentiation Primary Response 88–Cyclin D1 Signaling in Breast Cancer Cells Regulates Toll-Like Receptor 3-Mediated Cell Proliferation
    Article Snippet: Briefly, 40 × 104 cells were seeded on a coverslip in a 35-mm culture dish in complete media. .. Four hours before the addition of the TLR3 ligand, the MyD88 inhibitor was added and incubated for 24 h. For TLR3 surface expression, cells were fixed and allowed to bind with TLR 3 antibody (Invitrogen-PA5-29619) and Alexa 594-conjugated secondary anti-rabbit goat antibody (Invitrogen-A11012). .. For IL-6 expression, cells were fixed, permeabilized, and incubated with primary IL-6 antibody (Invitrogen-AMC0862) and Alexa 488-conjugated anti-mouse goat antibody (Invitrogen-A11001) and mounted with Vecta Shield-DAPI to counterstain the nuclei and were observed under fluorescence microscope (Leica DMI 6000B).

    Immunohistochemistry:

    Article Title: Sensory neurons contacting the cerebrospinal fluid require the Reissner fiber to detect spinal curvature in vivo
    Article Snippet: The following primary antibodies were used for in toto immunohistochemistry: rabbit anti-Reissner fiber (1:200, polyclonal, custom-made) [ , ], rabbit anti-Pkd2l1 (1:200, custom-made, Sternberg et al., 2018), mouse anti-tagRFP (1:500, MA515257, Thermo Fischer Scientific, Waltham, Massachusetts, USA), mouse anti-ZO-1 (1:200, 339100, Invitrogen) and chicken anti-GFP (1:500, ab13970, Abcam, Cambridge, England). .. The following primary antibodies were used for in toto immunohistochemistry: rabbit anti-Reissner fiber (1:200, polyclonal, custom-made) [ , ], rabbit anti-Pkd2l1 (1:200, custom-made, Sternberg et al., 2018), mouse anti-tagRFP (1:500, MA515257, Thermo Fischer Scientific, Waltham, Massachusetts, USA), mouse anti-ZO-1 (1:200, 339100, Invitrogen) and chicken anti-GFP (1:500, ab13970, Abcam, Cambridge, England). .. The following secondary antibodies were used at 1:500: Alexa Fluor-568 goat anti-rabbit IgG A11036, Alexa Fluor-488 donkey anti-rabbit A21206, Alexa Fluor-647 goat anti-rabbit IgG A21244, Alexa Fluor-555 goat anti-mouse IgG1 A21127, Alexa Fluor-568 goat anti-mouse A11004, Alexa Fluor-488 goat anti chicken IgG A11039 (Thermo Fischer Scientific, Waltham, Massachusetts, USA).

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    Thermo Fisher rabbit anti alexa 488 antibody
    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for <t>Alexa</t> 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.
    Rabbit Anti Alexa 488 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti alexa 488 antibody/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti alexa 488 antibody - by Bioz Stars, 2021-04
    97/100 stars
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    97
    Thermo Fisher goat anti rabbit alexa fluor 546
    Flagella of both EcN and EcN BGs were identified using immunohistochemistry and immunoelectron microscopy. Notes: ( A ) EcN (pBGKB-MOMP, pGLysivb) bacteria, transmitted light; ( B ) flagella of EcN (pBGKB-MOMP, pGLysivb) are localized and labeled with <t>Alexa</t> <t>Fluor</t> 546 (white arrowheads); ( C ) MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs, transmitted light; ( D ) flagella of MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs are localized and labeled with Alexa Fluor 546 (white arrowheads); ( E ) TEM image of EcN (pBGKB-MOMP, pGLysivb) bacteria, showing negatively contrasted flagella labeled with 25 nm colloidal gold; ( F ) high-magnification TEM image of EcN (pBGKB-MOMP, pGLysivb) bacteria, showing negatively contrasted flagella labeled with 6 nm colloidal gold; ( G ) lower magnification and ( H ) high-magnification TEM image of MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs showing negatively contrasted flagella labeled with 12 nm colloidal gold. Scale bars: ( A – D ) 5 µm; ( E ) 2 µm; and ( F – H ) 500 nm. Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; TEM, transmission electron microscope.
    Goat Anti Rabbit Alexa Fluor 546, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti rabbit alexa fluor 546/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti rabbit alexa fluor 546 - by Bioz Stars, 2021-04
    97/100 stars
      Buy from Supplier

    Image Search Results


    Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Journal: Nature biotechnology

    Article Title: Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy

    doi: 10.1038/s41587-019-0073-7

    Figure Lengend Snippet: Acid-switching results in increased accumulation of ADCs in HER2-expressing tumor cells. Cells were incubated with 10 nM Alexa 488-labeled MMAE-conjugated antibody (WT, SG, YS or control hen egg lysozyme-specific antibody, C) for the indicated times at 37 °C, washed, incubated with 5 μg/ml Alexa 488-specific antibody and analyzed by flow cytometry. Mean fluorescence intensities (mean values of independent triplicate cell samples) for Alexa 488 fluorescence are shown. Error bars indicate SD. Statistically significant differences are indicated by * (unpaired two-tailed t -test). Two independent experiments were carried out with similar results.

    Article Snippet: Samples were treated with 5 μg/ml rabbit anti-Alexa 488 antibody for 30 minutes on ice to quench surface Alexa 488 fluorescence, and subsequently fixed at room temperature with 3.4% (w/v) paraformaldehyde plus 0.025% (v/v) glutaraldehyde.

    Techniques: Expressing, Incubation, Labeling, Flow Cytometry, Cytometry, Fluorescence, Two Tailed Test

    Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

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

    Article Title: Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1

    doi: 10.1073/pnas.2536800100

    Figure Lengend Snippet: Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

    Article Snippet: Stainings were visualized with Alexa 488-conjugated secondary anti-goat antibodies (Molecular Probes) and nuclei were counterstained with Hoechst 33342 (Molecular Probes).

    Techniques: Expressing, Mouse Assay, Injection, Staining, Immunofluorescence

    Flagella of both EcN and EcN BGs were identified using immunohistochemistry and immunoelectron microscopy. Notes: ( A ) EcN (pBGKB-MOMP, pGLysivb) bacteria, transmitted light; ( B ) flagella of EcN (pBGKB-MOMP, pGLysivb) are localized and labeled with Alexa Fluor 546 (white arrowheads); ( C ) MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs, transmitted light; ( D ) flagella of MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs are localized and labeled with Alexa Fluor 546 (white arrowheads); ( E ) TEM image of EcN (pBGKB-MOMP, pGLysivb) bacteria, showing negatively contrasted flagella labeled with 25 nm colloidal gold; ( F ) high-magnification TEM image of EcN (pBGKB-MOMP, pGLysivb) bacteria, showing negatively contrasted flagella labeled with 6 nm colloidal gold; ( G ) lower magnification and ( H ) high-magnification TEM image of MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs showing negatively contrasted flagella labeled with 12 nm colloidal gold. Scale bars: ( A – D ) 5 µm; ( E ) 2 µm; and ( F – H ) 500 nm. Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; TEM, transmission electron microscope.

    Journal: Drug Design, Development and Therapy

    Article Title: Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface

    doi: 10.2147/DDDT.S84370

    Figure Lengend Snippet: Flagella of both EcN and EcN BGs were identified using immunohistochemistry and immunoelectron microscopy. Notes: ( A ) EcN (pBGKB-MOMP, pGLysivb) bacteria, transmitted light; ( B ) flagella of EcN (pBGKB-MOMP, pGLysivb) are localized and labeled with Alexa Fluor 546 (white arrowheads); ( C ) MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs, transmitted light; ( D ) flagella of MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs are localized and labeled with Alexa Fluor 546 (white arrowheads); ( E ) TEM image of EcN (pBGKB-MOMP, pGLysivb) bacteria, showing negatively contrasted flagella labeled with 25 nm colloidal gold; ( F ) high-magnification TEM image of EcN (pBGKB-MOMP, pGLysivb) bacteria, showing negatively contrasted flagella labeled with 6 nm colloidal gold; ( G ) lower magnification and ( H ) high-magnification TEM image of MOMP-EcN (pBGKB-MOMP, pGLysivb) BGs showing negatively contrasted flagella labeled with 12 nm colloidal gold. Scale bars: ( A – D ) 5 µm; ( E ) 2 µm; and ( F – H ) 500 nm. Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; TEM, transmission electron microscope.

    Article Snippet: The secondary antibodies of donkey anti-mouse Alexa Fluor 488 (Invitrogen, Life Technologies) and goat anti-rabbit Alexa Fluor 546 (Thermo Fisher Scientific) diluted to 1:500 in antibody diluent were applied simultaneously for 20 minutes at 37°C.

    Techniques: Immunohistochemistry, Immuno-Electron Microscopy, Labeling, Transmission Electron Microscopy, Transmission Assay, Microscopy

    Microscopic confirmation of BGs expressing recombinant foreign chlamydial antigens. Notes: ( A ) Whole EcN BGs (pBGKB-MOMP, pGLysivb) bacteria, transmitted light; ( B ) whole EcN (pBGKB-MOMP, pGLysivb) are localized and labeled with Alexa Fluor 546 ( Escherichia coli outer membrane) and Alexa Fluor 488 (c-Myc-MOMP); ( C ) semi-thin sections of EcN BGs (pBGKB-MOMP, pGLysivb) are stained with toluidine blue, transmitted light; ( D ) expressed proteins of EcN BGs (pBGKB-MOMP, pGLysivb) are localized and labeled on semi-thin sections with Alexa Fluor 546 ( E. coli outer membrane) and Alexa Fluor 488 (c-Myc-MOMP); ( E ) TEM image of EcN (pBGKB-N-PmpC, pGLysivb) bacteria; ( F ) TEM image of EcN BGs (pBGKB-N-PmpC, pGLysivb) bacteria; ( G ) TEM image of EcN BGs (pBGKB-N-PmpC, pGLysivb) bacteria; and ( H ) TEM image of N-PmpC-EcN BGs (pBGKB-N-PmpC, pGLysivb) showing c-Myc-N-PmpC expressed protein, labeled with 12 nm colloidal gold. The arrows in ( F ) and ( H ) show areas labeled with 10 nm colloidal gold particles, indicating N-PmpC antigen co-expressed with c-Myc protein, in the bacterial cell ( F ) and also in the BG ( H ). Scale bars: ( A – D ) 5 µm; ( E ) 1 µm; ( F ) 200 nm; ( G ) 1 µm; and ( H ) 500 nm. Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; N-PmpC, N-terminal part of polymorphic membrane protein C; TEM, transmission electron microscope.

    Journal: Drug Design, Development and Therapy

    Article Title: Escherichia coli Nissle 1917 bacterial ghosts retain crucial surface properties and express chlamydial antigen: an imaging study of a delivery system for the ocular surface

    doi: 10.2147/DDDT.S84370

    Figure Lengend Snippet: Microscopic confirmation of BGs expressing recombinant foreign chlamydial antigens. Notes: ( A ) Whole EcN BGs (pBGKB-MOMP, pGLysivb) bacteria, transmitted light; ( B ) whole EcN (pBGKB-MOMP, pGLysivb) are localized and labeled with Alexa Fluor 546 ( Escherichia coli outer membrane) and Alexa Fluor 488 (c-Myc-MOMP); ( C ) semi-thin sections of EcN BGs (pBGKB-MOMP, pGLysivb) are stained with toluidine blue, transmitted light; ( D ) expressed proteins of EcN BGs (pBGKB-MOMP, pGLysivb) are localized and labeled on semi-thin sections with Alexa Fluor 546 ( E. coli outer membrane) and Alexa Fluor 488 (c-Myc-MOMP); ( E ) TEM image of EcN (pBGKB-N-PmpC, pGLysivb) bacteria; ( F ) TEM image of EcN BGs (pBGKB-N-PmpC, pGLysivb) bacteria; ( G ) TEM image of EcN BGs (pBGKB-N-PmpC, pGLysivb) bacteria; and ( H ) TEM image of N-PmpC-EcN BGs (pBGKB-N-PmpC, pGLysivb) showing c-Myc-N-PmpC expressed protein, labeled with 12 nm colloidal gold. The arrows in ( F ) and ( H ) show areas labeled with 10 nm colloidal gold particles, indicating N-PmpC antigen co-expressed with c-Myc protein, in the bacterial cell ( F ) and also in the BG ( H ). Scale bars: ( A – D ) 5 µm; ( E ) 1 µm; ( F ) 200 nm; ( G ) 1 µm; and ( H ) 500 nm. Abbreviations: BGs, bacterial ghosts; EcN, Escherichia coli strain Nissle 1917; MOMP, major outer membrane protein; N-PmpC, N-terminal part of polymorphic membrane protein C; TEM, transmission electron microscope.

    Article Snippet: The secondary antibodies of donkey anti-mouse Alexa Fluor 488 (Invitrogen, Life Technologies) and goat anti-rabbit Alexa Fluor 546 (Thermo Fisher Scientific) diluted to 1:500 in antibody diluent were applied simultaneously for 20 minutes at 37°C.

    Techniques: Expressing, Recombinant, Labeling, Staining, Transmission Electron Microscopy, Transmission Assay, Microscopy

    Signalling-defective DDR1 mutants bind triple-helical DDR1 selective peptide but do not phosphorylate with peptide stimulation. ( A ) COS-7 cells transiently expressing wild-type DDR1 or the indicated DDR1 mutant were incubated with or without a biotinylated DDR-selective collagen-mimetic peptide for 60 minutes on ice, followed by incubation with anti-DDR1 mAb 7A9 on ice. Cells were then fixed and incubated with Alexa Fluor-488 goat-anti-mouse IgG and Alexa Fluor-546 conjugated streptavidin. Cells were imaged by widefield microscopy. The graph shows mean fluorescence intensity, normalised to respective DDR1 expression levels. N = 27–31 fields of view from 3 independent experiments. Scale bar, 20 μm. ( B ) HEK293 transiently expressing wild-type DDR1 or the indicated DDR1 mutant were stimulated with collagen I (C), or with DDR-selective collagen-mimetic peptide (P) for 60 minutes at 37 °C or were left unstimulated. Cell lysates were analysed by Western blot using an Ab against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). NS, no significance; *p

    Journal: Scientific Reports

    Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

    doi: 10.1038/s41598-019-53176-4

    Figure Lengend Snippet: Signalling-defective DDR1 mutants bind triple-helical DDR1 selective peptide but do not phosphorylate with peptide stimulation. ( A ) COS-7 cells transiently expressing wild-type DDR1 or the indicated DDR1 mutant were incubated with or without a biotinylated DDR-selective collagen-mimetic peptide for 60 minutes on ice, followed by incubation with anti-DDR1 mAb 7A9 on ice. Cells were then fixed and incubated with Alexa Fluor-488 goat-anti-mouse IgG and Alexa Fluor-546 conjugated streptavidin. Cells were imaged by widefield microscopy. The graph shows mean fluorescence intensity, normalised to respective DDR1 expression levels. N = 27–31 fields of view from 3 independent experiments. Scale bar, 20 μm. ( B ) HEK293 transiently expressing wild-type DDR1 or the indicated DDR1 mutant were stimulated with collagen I (C), or with DDR-selective collagen-mimetic peptide (P) for 60 minutes at 37 °C or were left unstimulated. Cell lysates were analysed by Western blot using an Ab against phosphorylated Tyr-513 (anti-pY). The blot was stripped and re-probed with anti-DDR1. The positions of molecular mass markers are indicated on the left in kDa. The bar chart shows the densitometry analysis of pY513 band intensities after normalization to total DDR1. Each value is a percentage of the sum of all the pY513/DDR1 signals on the blot. The graph shows mean band intensities + SEM (N = 3). NS, no significance; *p

    Article Snippet: Secondary Abs were as follows: goat anti-rabbit Ig-horseradish peroxidase conjugated (P0448, DAKO A/S, Denmark); sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK); goat anti-mouse IgG Alexa Flour-488 (Invitrogen); goat anti-mouse IgG1 Alexa Fluor-488 (Invitrogen); goat anti-rabbit Alexa Fluor-546 (Invitrogen); goat anti-mouse IgG2b Alexa Fluor-555 (Invitrogen); goat anti-rabbit IgG Alexa Fluor-647 (Invitrogen); goat anti-mouse IgG FITC-conjugated (F-9006, Sigma).

    Techniques: Expressing, Mutagenesis, Incubation, Microscopy, Fluorescence, Western Blot

    Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. ( A ) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. ( B , C ) COS-7 cells transiently expressing DDR1-SNAP were incubated with SNAP-Surface Alexa Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes ( B ) or for 5, 10, or 60 minutes ( C ) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining ( B ), or fixed and mounted ( C ). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices ( B ) or from a single slice ( A , C ). White boxes indicate corresponding areas shown at higher magnification in lower images ( B ). Scale bars, 5 μm ( A ), 30 μm (upper image in B), 2 μm (enlarged images in B) or 3 μm ( C ). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal ( B ). At least 10 cells were imaged for each condition.

    Journal: Scientific Reports

    Article Title: DDR1 autophosphorylation is a result of aggregation into dense clusters

    doi: 10.1038/s41598-019-53176-4

    Figure Lengend Snippet: Aggregated and phosphorylated DDR1 is present in the double walled anti-DDR1 structures. ( A ) COS-7 cells transiently expressing DDR1 were stimulated with collagen I for 60 minutes at 37 °C, then incubated on ice with anti-DDR1 mAb 7A9 and anti-collagen I mAb, before fixation, permeabilisation and immunostaining for phospho-tyrosine 513 (pY-DDR1). Intensity of the three stains was measured across the three lines shown (with a line width of 200 nm), the data were normalised so that the lowest and highest value from each stain was 0 and 100 A.U. ( B , C ) COS-7 cells transiently expressing DDR1-SNAP were incubated with SNAP-Surface Alexa Fluor-546 for 60 minutes at 37 °C, then stimulated with collagen I for 60 minutes ( B ) or for 5, 10, or 60 minutes ( C ) at 37 °C. Cells were then incubated on ice with anti-DDR1 mAb 5D5, before fixation, and secondary Ab staining ( B ), or fixed and mounted ( C ). 3D-SIM images were acquired using a Zeiss ELRYA microscope. Images are from a maximum intensity projection of all 15 slices ( B ) or from a single slice ( A , C ). White boxes indicate corresponding areas shown at higher magnification in lower images ( B ). Scale bars, 5 μm ( A ), 30 μm (upper image in B), 2 μm (enlarged images in B) or 3 μm ( C ). White arrows indicate examples of anti-DDR1 mAb binding at the edges of aggregated DDR1-SNAP signal ( B ). At least 10 cells were imaged for each condition.

    Article Snippet: Secondary Abs were as follows: goat anti-rabbit Ig-horseradish peroxidase conjugated (P0448, DAKO A/S, Denmark); sheep anti-mouse Ig-horseradish peroxidase (Amersham Biosciences, Little Chalfont, UK); goat anti-mouse IgG Alexa Flour-488 (Invitrogen); goat anti-mouse IgG1 Alexa Fluor-488 (Invitrogen); goat anti-rabbit Alexa Fluor-546 (Invitrogen); goat anti-mouse IgG2b Alexa Fluor-555 (Invitrogen); goat anti-rabbit IgG Alexa Fluor-647 (Invitrogen); goat anti-mouse IgG FITC-conjugated (F-9006, Sigma).

    Techniques: Expressing, Incubation, Immunostaining, Staining, Microscopy, Binding Assay