biotinylated secondary antibodies  (Vector Laboratories)


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    Name:
    Biotinylated Anti Avidin Antibody
    Description:
    Biotinylated Anti Avidin has been widely used as an amplifying reagent in immunohistochemistry in situ hybridization microarray assays ELISAs blots and many other applications The capability of binding avidin via either biotin binding sites or through antigen binding sites makes this biotinylated antibody unique Our antibodies to avidin are produced in goats using our highly purified avidin and isolated by affinity chromatography Anti Avidin does not bind streptavidin and Anti Streptavidin does not recognize avidin These antibodies provide opportunities to significantly amplify signals in many applications
    Catalog Number:
    ba-0300
    Price:
    None
    Category:
    Antibodies
    Size:
    0 5 mg
    Host:
    Goat
    Buy from Supplier


    Structured Review

    Vector Laboratories biotinylated secondary antibodies
    Biotinylated Anti Avidin Antibody
    Biotinylated Anti Avidin has been widely used as an amplifying reagent in immunohistochemistry in situ hybridization microarray assays ELISAs blots and many other applications The capability of binding avidin via either biotin binding sites or through antigen binding sites makes this biotinylated antibody unique Our antibodies to avidin are produced in goats using our highly purified avidin and isolated by affinity chromatography Anti Avidin does not bind streptavidin and Anti Streptavidin does not recognize avidin These antibodies provide opportunities to significantly amplify signals in many applications
    https://www.bioz.com/result/biotinylated secondary antibodies/product/Vector Laboratories
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated secondary antibodies - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection"

    Article Title: Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection

    Journal: Scientific Reports

    doi: 10.1038/srep09534

    CD31 expression detected with different detection systems. Paraformaldehyde fixed, paraffin embedded 5 μm sections of E13.5 mouse embryo were stained with CD31 primary antibody (see Methods for details) and nuclei were stained with DAPI. Panel A shows detection with biotinylated goat anti-rat secondary antibodies followed by Streptavidin-HRP and TSA- Alexa488 amplification. Panel B shows detection with goat anti-rat secondary antibodies directly conjugated with Alexa488. Panel C shows detection with biotinylated goat anti-rat antibodies followed by Streptavidin-Alexa488. Panel D shows staining with isotype control IgG detected with biotinylated goat anti-rat antibodies, followed by Streptavidin-HRP and TSA-Alexa488. The inset in each panel represents a closer view of the indicated region, with CD31 signal showing in grayscale. White arrows point to autofluorescence emitted by the eurethrocytes, which persists in all staining methods.
    Figure Legend Snippet: CD31 expression detected with different detection systems. Paraformaldehyde fixed, paraffin embedded 5 μm sections of E13.5 mouse embryo were stained with CD31 primary antibody (see Methods for details) and nuclei were stained with DAPI. Panel A shows detection with biotinylated goat anti-rat secondary antibodies followed by Streptavidin-HRP and TSA- Alexa488 amplification. Panel B shows detection with goat anti-rat secondary antibodies directly conjugated with Alexa488. Panel C shows detection with biotinylated goat anti-rat antibodies followed by Streptavidin-Alexa488. Panel D shows staining with isotype control IgG detected with biotinylated goat anti-rat antibodies, followed by Streptavidin-HRP and TSA-Alexa488. The inset in each panel represents a closer view of the indicated region, with CD31 signal showing in grayscale. White arrows point to autofluorescence emitted by the eurethrocytes, which persists in all staining methods.

    Techniques Used: Expressing, Staining, Amplification

    2) Product Images from "Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection"

    Article Title: Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection

    Journal: Scientific Reports

    doi: 10.1038/srep09534

    CD31 expression detected with different detection systems. Paraformaldehyde fixed, paraffin embedded 5 μm sections of E13.5 mouse embryo were stained with CD31 primary antibody (see Methods for details) and nuclei were stained with DAPI. Panel A shows detection with biotinylated goat anti-rat secondary antibodies followed by Streptavidin-HRP and TSA- Alexa488 amplification. Panel B shows detection with goat anti-rat secondary antibodies directly conjugated with Alexa488. Panel C shows detection with biotinylated goat anti-rat antibodies followed by Streptavidin-Alexa488. Panel D shows staining with isotype control IgG detected with biotinylated goat anti-rat antibodies, followed by Streptavidin-HRP and TSA-Alexa488. The inset in each panel represents a closer view of the indicated region, with CD31 signal showing in grayscale. White arrows point to autofluorescence emitted by the eurethrocytes, which persists in all staining methods.
    Figure Legend Snippet: CD31 expression detected with different detection systems. Paraformaldehyde fixed, paraffin embedded 5 μm sections of E13.5 mouse embryo were stained with CD31 primary antibody (see Methods for details) and nuclei were stained with DAPI. Panel A shows detection with biotinylated goat anti-rat secondary antibodies followed by Streptavidin-HRP and TSA- Alexa488 amplification. Panel B shows detection with goat anti-rat secondary antibodies directly conjugated with Alexa488. Panel C shows detection with biotinylated goat anti-rat antibodies followed by Streptavidin-Alexa488. Panel D shows staining with isotype control IgG detected with biotinylated goat anti-rat antibodies, followed by Streptavidin-HRP and TSA-Alexa488. The inset in each panel represents a closer view of the indicated region, with CD31 signal showing in grayscale. White arrows point to autofluorescence emitted by the eurethrocytes, which persists in all staining methods.

    Techniques Used: Expressing, Staining, Amplification

    Related Articles

    Staining:

    Article Title: Increased Epitope-Specific CD8+ T Cells Prevent Murine Coronavirus Spread to the Spinal Cord and Subsequent Demyelination
    Article Snippet: The right halves of brains and entire spinal cords from animals sacrificed at days 3, 5, and 7 p.i. were fixed in formalin, embedded in paraffin, sectioned, and stained for viral antigen or inflammation. .. Antigen staining was performed by the avidin-biotin-immunoperoxidase technique (Vector Laboratories, Burlingame, Calif.) by using diaminobenzidine tetrahydrochloride as the substrate and a 1:20 dilution of rabbit antinucleocapsid monoclonal antibody (kindly provided by Julian Leibowitz). .. Hematoxylin-and-eosin (H-and-E) staining for inflammation was carried out, and analysis was performed in a blinded manner by a neuropathologist.

    Article Title: Physiological Modulation of Intestinal Motility by Enteric Dopaminergic Neurons and the D2 Receptor: Analysis of Dopamine Receptor Expression, Location, Development, and Function in Wild-Type and Knock-Out Mice
    Article Snippet: Briefly, preparations were incubated (30 min) with 10% normal serum (of the species in which the secondary antibodies were raised) to inhibit nonspecific staining. .. The blocked tissue was then incubated overnight with primary antibodies, washed with PBS, and exposed with intervening washes in PBS to the biotinylated secondary antibodies (2 h), 0.3% H2 O2 in 0.3% blocking sera (5 min), Elite ABC reagent (1 h; Vector Laboratories), and 3′,5′-diaminobenzidine solution (2–10 min) until suitable staining developed. .. As a control for the specificity of antibodies to the D2 and D3 receptors, attempts were made to immunostain the corresponding receptors in the gut of knock-out animals lacking these receptors.

    Avidin-Biotin Assay:

    Article Title: Increased Epitope-Specific CD8+ T Cells Prevent Murine Coronavirus Spread to the Spinal Cord and Subsequent Demyelination
    Article Snippet: The right halves of brains and entire spinal cords from animals sacrificed at days 3, 5, and 7 p.i. were fixed in formalin, embedded in paraffin, sectioned, and stained for viral antigen or inflammation. .. Antigen staining was performed by the avidin-biotin-immunoperoxidase technique (Vector Laboratories, Burlingame, Calif.) by using diaminobenzidine tetrahydrochloride as the substrate and a 1:20 dilution of rabbit antinucleocapsid monoclonal antibody (kindly provided by Julian Leibowitz). .. Hematoxylin-and-eosin (H-and-E) staining for inflammation was carried out, and analysis was performed in a blinded manner by a neuropathologist.

    Article Title: Inflammatory Responses Are Not Sufficient to Cause Delayed Neuronal Death in ATP-Induced Acute Brain Injury
    Article Snippet: Sections were incubated overnight at room temperature with primary antibodies against tyrosine hydroxylase (TH; 1∶2000 dilution; Pelfreeze Biologicals, Rogers, AR), ionized calcium binding adaptor molecule 1 (Iba-1; 1∶1000; Wako Pure Chemical Industries, Osaka, Japan), myeloperoxidase (MPO; 1∶1000; Dako, Glostrup, Denmark), CD45 (1∶200; AbD Serotec, Oxford, UK), CD11b (OX-42; 1∶200; Serotec, Oxford, UK), Ki-67 (1∶100; Abcam, Cambridge, UK), interleukin-1β (IL-1β; 1∶200; R & D systems, MN), inducible nitric oxide synthase (iNOS; 1∶200; Abcam, Cambridge, UK), CD68 (1∶200; AbD Serotec, oxford, UK) or Neuronal Nuclei (NeuN; 1: 300; Chemicon, CA, USA). .. Following rinsing in PBST, sections were incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) for 1 h and the avidin/biotin system (Vector Laboratories, Burlingame, CA) for 1 h and visualized using DAB solution (0.05% DAB and 0.003% hydrogen peroxide in 0.1 M PB). .. For double-labeling with TH and Iba-1, sections were stained with TH antibody following the above procedure, and visualized with DAB (brown product).

    Incubation:

    Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression
    Article Snippet: Immunohistochemistry Formalin-fixed, paraffin-embedded sections were deparaffinized in xylene, rehydrated in alcohol, and processed as follows: Sections were incubated with target retrieval solution (Dako) in a steamer for 45 min followed by 3% hydrogen peroxide solution for 10 min and protein block (Dako) for 20 min at room temperature. .. Sections were incubated overnight in a humid chamber at 4°C with antibody against Ki-67 purchased from Cell signaling technology company (Clone [D2H10], Cat. #9027, 1:1600 dilution) followed by biotinylated secondary antibody (Vector laboratories) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (Dako), and sections were counterstained with hematoxylin before mounting. .. Micrographs of stained sections were taken using a Leica DMIL LED microscope with Amscope camera and acquisition software.

    Article Title: Anti-GRP78 autoantibodies induce endothelial cell activation and accelerate the development of atherosclerotic lesions
    Article Snippet: Aortic sections (4-μm thick) were deparaffinized, and the endogenous peroxidase activity was blocked with 0.5% H2 O2 in methanol for 10 minutes, as described previously ( ). .. Following antigen retrieval where necessary, sections were blocked with 5% normal serum, and incubated with the primary antibody for 1 hour, followed by biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:500, and streptavidin HRP (Vector Laboratories) diluted 1:50. .. Sections were developed in Nova Red peroxidase substrate (Vector Laboratories) and counterstained with hematoxylin.

    Article Title: Y-cell receptive field and collicular projection of parasol ganglion cells in macaque monkey retina
    Article Snippet: In some retinas we alternatively improved photostaining by an additional immunohistochemical step using biotinylated anti-rhodamine prior to HRP processing. .. Following fixation, retinas were incubated in 0.1% Triton X-100 in PBS (0.1 M, pH 7.4) containing biotinylated anti-rhodamine (C# BA-0605, Vector Labs) at a concentration of 1:100 for 36 hr at room temperature, then washed in phosphate buffer overnight. .. Retinas were then processed for HRP histochemistry as described above using the Vector avidin-biotin-HRP complex and DAB.

    Article Title: Physiological Modulation of Intestinal Motility by Enteric Dopaminergic Neurons and the D2 Receptor: Analysis of Dopamine Receptor Expression, Location, Development, and Function in Wild-Type and Knock-Out Mice
    Article Snippet: Briefly, preparations were incubated (30 min) with 10% normal serum (of the species in which the secondary antibodies were raised) to inhibit nonspecific staining. .. The blocked tissue was then incubated overnight with primary antibodies, washed with PBS, and exposed with intervening washes in PBS to the biotinylated secondary antibodies (2 h), 0.3% H2 O2 in 0.3% blocking sera (5 min), Elite ABC reagent (1 h; Vector Laboratories), and 3′,5′-diaminobenzidine solution (2–10 min) until suitable staining developed. .. As a control for the specificity of antibodies to the D2 and D3 receptors, attempts were made to immunostain the corresponding receptors in the gut of knock-out animals lacking these receptors.

    Article Title: Inflammatory Responses Are Not Sufficient to Cause Delayed Neuronal Death in ATP-Induced Acute Brain Injury
    Article Snippet: Sections were incubated overnight at room temperature with primary antibodies against tyrosine hydroxylase (TH; 1∶2000 dilution; Pelfreeze Biologicals, Rogers, AR), ionized calcium binding adaptor molecule 1 (Iba-1; 1∶1000; Wako Pure Chemical Industries, Osaka, Japan), myeloperoxidase (MPO; 1∶1000; Dako, Glostrup, Denmark), CD45 (1∶200; AbD Serotec, Oxford, UK), CD11b (OX-42; 1∶200; Serotec, Oxford, UK), Ki-67 (1∶100; Abcam, Cambridge, UK), interleukin-1β (IL-1β; 1∶200; R & D systems, MN), inducible nitric oxide synthase (iNOS; 1∶200; Abcam, Cambridge, UK), CD68 (1∶200; AbD Serotec, oxford, UK) or Neuronal Nuclei (NeuN; 1: 300; Chemicon, CA, USA). .. Following rinsing in PBST, sections were incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) for 1 h and the avidin/biotin system (Vector Laboratories, Burlingame, CA) for 1 h and visualized using DAB solution (0.05% DAB and 0.003% hydrogen peroxide in 0.1 M PB). .. For double-labeling with TH and Iba-1, sections were stained with TH antibody following the above procedure, and visualized with DAB (brown product).

    Article Title: Region-specific astrogliosis: differential vessel formation contributes to different patterns of astrogliosis in the cortex and striatum
    Article Snippet: After incubation with 1% bovine serum albumin (BSA) in PBS, the sections were incubated overnight at 4 °C with primary antibodies (Table ). .. The sections were rinsed three times with PBS and incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA). .. Immunoreactive proteins were visualized using an avidin-biotin-peroxidase-DAB solution (0.05% DAB and 0.003% H2 O2 in 0.1 M PB) according to the manufacturer’s instructions.

    Concentration Assay:

    Article Title: Y-cell receptive field and collicular projection of parasol ganglion cells in macaque monkey retina
    Article Snippet: In some retinas we alternatively improved photostaining by an additional immunohistochemical step using biotinylated anti-rhodamine prior to HRP processing. .. Following fixation, retinas were incubated in 0.1% Triton X-100 in PBS (0.1 M, pH 7.4) containing biotinylated anti-rhodamine (C# BA-0605, Vector Labs) at a concentration of 1:100 for 36 hr at room temperature, then washed in phosphate buffer overnight. .. Retinas were then processed for HRP histochemistry as described above using the Vector avidin-biotin-HRP complex and DAB.

    Blocking Assay:

    Article Title: Physiological Modulation of Intestinal Motility by Enteric Dopaminergic Neurons and the D2 Receptor: Analysis of Dopamine Receptor Expression, Location, Development, and Function in Wild-Type and Knock-Out Mice
    Article Snippet: Briefly, preparations were incubated (30 min) with 10% normal serum (of the species in which the secondary antibodies were raised) to inhibit nonspecific staining. .. The blocked tissue was then incubated overnight with primary antibodies, washed with PBS, and exposed with intervening washes in PBS to the biotinylated secondary antibodies (2 h), 0.3% H2 O2 in 0.3% blocking sera (5 min), Elite ABC reagent (1 h; Vector Laboratories), and 3′,5′-diaminobenzidine solution (2–10 min) until suitable staining developed. .. As a control for the specificity of antibodies to the D2 and D3 receptors, attempts were made to immunostain the corresponding receptors in the gut of knock-out animals lacking these receptors.

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    Vector Laboratories biotinylated secondary antibody reagent
    Functional activities of gC1qR MAbs 74.5.2 and 60.11 in rat serum. <t>Biotinylated,</t> clfA -positive bacteria were suspended (10% cell suspension) in selected serum samples from animals treated with gC1qR MAbs. Samples were applied to fibrinogen-coated microtiter
    Biotinylated Secondary Antibody Reagent, supplied by Vector Laboratories, 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/biotinylated secondary antibody reagent/product/Vector Laboratories
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated secondary antibody reagent - by Bioz Stars, 2021-03
    99/100 stars
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    Functional activities of gC1qR MAbs 74.5.2 and 60.11 in rat serum. Biotinylated, clfA -positive bacteria were suspended (10% cell suspension) in selected serum samples from animals treated with gC1qR MAbs. Samples were applied to fibrinogen-coated microtiter

    Journal:

    Article Title: gC1qR/p33 Blockade Reduces Staphylococcus aureus Colonization of Target Tissues in an Animal Model of Infective Endocarditis

    doi: 10.1128/IAI.01794-05

    Figure Lengend Snippet: Functional activities of gC1qR MAbs 74.5.2 and 60.11 in rat serum. Biotinylated, clfA -positive bacteria were suspended (10% cell suspension) in selected serum samples from animals treated with gC1qR MAbs. Samples were applied to fibrinogen-coated microtiter

    Article Snippet: Antibody was detected by using a biotinylated secondary antibody reagent at room temperature for 2 h (Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA) ( ).

    Techniques: Functional Assay

    Death of dopaminergic neurons and microglia in the SNpc induced by ATP. ( A ) ATP (100 nmol in 2 µl PBS) or PBS (2 µl) was unilaterally injected into SNpc (*, injection sites), and brains were obtained after 3 h. Brain sections (30 µm thickness) of the midbrain including the entire SN were prepared, every sixth serial section selected and stained with TH (upper panel) and Iba-1 (lower panel) antibodies, and visualized with biotin-conjugated secondary antibodies and enzymatic detection with the avidin/biotin system unless indicated. At 100 nmol, mild neuronal and microglial damage occurred, thus, 100 nmol ATP was employed for in vivo experiments in this study. Photographs of the most damaged sections were obtained. The contralateral side (contra) and PBS-injected rat brain sections were used as control. ( B ) Serial sections obtained at 3 h were labeled with TH (left panel), Iba-1 (middle panel), and TH/Iba-1 (right panel) antibodies. For visualization of the double-labeling, color reactions using DAB (for TH) and DAB/nickel sulfate (for Iba-1) were applied. Dotted lines indicated damage areas. ( C ) Brain tissue obtained 3 h post ATP treatment was subjected to electron microscopy, as described in “ Materials and Methods ”. Nuclei of neuron (N, white arrow), astrocytes (A, white arrowhead), and microglia (M, black arrow) were shown in intact rat brain whereas cellular structures were severely disrupted in ATP-injected brain. Data in this study are representative of at least 5 animals. Scale bars, 200 µm (A); 100 µm (B); 5 µm (C).

    Journal: PLoS ONE

    Article Title: Inflammatory Responses Are Not Sufficient to Cause Delayed Neuronal Death in ATP-Induced Acute Brain Injury

    doi: 10.1371/journal.pone.0013756

    Figure Lengend Snippet: Death of dopaminergic neurons and microglia in the SNpc induced by ATP. ( A ) ATP (100 nmol in 2 µl PBS) or PBS (2 µl) was unilaterally injected into SNpc (*, injection sites), and brains were obtained after 3 h. Brain sections (30 µm thickness) of the midbrain including the entire SN were prepared, every sixth serial section selected and stained with TH (upper panel) and Iba-1 (lower panel) antibodies, and visualized with biotin-conjugated secondary antibodies and enzymatic detection with the avidin/biotin system unless indicated. At 100 nmol, mild neuronal and microglial damage occurred, thus, 100 nmol ATP was employed for in vivo experiments in this study. Photographs of the most damaged sections were obtained. The contralateral side (contra) and PBS-injected rat brain sections were used as control. ( B ) Serial sections obtained at 3 h were labeled with TH (left panel), Iba-1 (middle panel), and TH/Iba-1 (right panel) antibodies. For visualization of the double-labeling, color reactions using DAB (for TH) and DAB/nickel sulfate (for Iba-1) were applied. Dotted lines indicated damage areas. ( C ) Brain tissue obtained 3 h post ATP treatment was subjected to electron microscopy, as described in “ Materials and Methods ”. Nuclei of neuron (N, white arrow), astrocytes (A, white arrowhead), and microglia (M, black arrow) were shown in intact rat brain whereas cellular structures were severely disrupted in ATP-injected brain. Data in this study are representative of at least 5 animals. Scale bars, 200 µm (A); 100 µm (B); 5 µm (C).

    Article Snippet: Following rinsing in PBST, sections were incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) for 1 h and the avidin/biotin system (Vector Laboratories, Burlingame, CA) for 1 h and visualized using DAB solution (0.05% DAB and 0.003% hydrogen peroxide in 0.1 M PB).

    Techniques: Injection, Staining, Avidin-Biotin Assay, In Vivo, Labeling, Electron Microscopy

    D 2 and D 3 immunocytochemistry were performed on the submucosal plexus of the ileum of CD-1 mice. The same tissue preparation from D 2 and D 3 KO mice was used as control. D 2 receptor immunoreactivity (IR) was revealed by a D 2 rabbit antibody and a donkey anti-rabbit Alexa 594 secondary antibody. D 3 receptor immunoreactivity was revealed by a D 3 goat antibody, a biotinylated donkey anti-goat secondary antibody, and streptavidin FITC. On the tissue of CD-1 mice, D 2 -immunoreactive products were present in the enteric neurons ( A ), and D 3 -immunoreactive products were also present in the enteric neurons ( B ). D 2 and D 3 immunoreactivities were colocalized in the same cell ( C ). However, D 2 immunoreactivity was not detected on the tissue of D 2 knock-out (KO) mice ( D ); no D 3 immunoreactivity was detected on the tissue of D 3 knock-out mouse ( E ). The arrows indicate the immunoreactive neurons. Scale bar: (in C ) 25 μm.

    Journal: The Journal of Neuroscience

    Article Title: Physiological Modulation of Intestinal Motility by Enteric Dopaminergic Neurons and the D2 Receptor: Analysis of Dopamine Receptor Expression, Location, Development, and Function in Wild-Type and Knock-Out Mice

    doi: 10.1523/JNEUROSCI.4720-05.2006

    Figure Lengend Snippet: D 2 and D 3 immunocytochemistry were performed on the submucosal plexus of the ileum of CD-1 mice. The same tissue preparation from D 2 and D 3 KO mice was used as control. D 2 receptor immunoreactivity (IR) was revealed by a D 2 rabbit antibody and a donkey anti-rabbit Alexa 594 secondary antibody. D 3 receptor immunoreactivity was revealed by a D 3 goat antibody, a biotinylated donkey anti-goat secondary antibody, and streptavidin FITC. On the tissue of CD-1 mice, D 2 -immunoreactive products were present in the enteric neurons ( A ), and D 3 -immunoreactive products were also present in the enteric neurons ( B ). D 2 and D 3 immunoreactivities were colocalized in the same cell ( C ). However, D 2 immunoreactivity was not detected on the tissue of D 2 knock-out (KO) mice ( D ); no D 3 immunoreactivity was detected on the tissue of D 3 knock-out mouse ( E ). The arrows indicate the immunoreactive neurons. Scale bar: (in C ) 25 μm.

    Article Snippet: The blocked tissue was then incubated overnight with primary antibodies, washed with PBS, and exposed with intervening washes in PBS to the biotinylated secondary antibodies (2 h), 0.3% H2 O2 in 0.3% blocking sera (5 min), Elite ABC reagent (1 h; Vector Laboratories), and 3′,5′-diaminobenzidine solution (2–10 min) until suitable staining developed.

    Techniques: Immunocytochemistry, Mouse Assay, Knock-Out

    PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense biotinylated probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.

    Journal: Nature cell biology

    Article Title: A regulated PNUTS mRNA to lncRNA splice switch mediates EMT and tumor progression

    doi: 10.1038/ncb3595

    Figure Lengend Snippet: PNUTS alternative splicing product is non-coding and interacts with miR-205 (a) Polysome fractionation experiment of A549 cells followed by RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression in each fraction. (b) RT-PCR analysis of PNUTS mRNA and lncRNA-PNUTS expression after the use of oligo-(dT) or random hexanucleotides as primers for initial reverse transcription reaction. (c) RT-PCR analysis of lncRNA-PNUTS expression in A549 cells. The total, cytoplasmic (Cyto.) and nuclear fractions are shown. PNUTS pre-RNA and PNUTS mRNA were used as endogenous controls to monitor the fractions purity. (d) Confocal microscopy imaging of subcellular localization of lncRNA-PNUTS using co-transfection of a MS2-tagged-RNA construct of lncRNA-PNUTS and a fused MS2-GFP protein construct. Scale bar: 5µM. (e) The exact copy numbers of lncRNA-PNUTS (basal levels or levels following activation by Actinomycin D treatment for 3h) and miR-205 were quantified with limiting-dilution qRT-PCR. Data are shown as mean ± s.d., n= 3 independent experiments per condition. Source data are available in Supplementary table 2 . (f) In silico prediction of MiR-205 binding sites located on lncRNA-PNUTS, obtained using the DIANA-microT web server. (g) Selective pull-down of either endogenous lncRNA-PNUTS or PNUTS-mRNA isoforms by using antisense biotinylated probes followed by miRNA-specific RT-PCR analysis to detect endogenously associated miR-205 with lncRNA-PNUTS in A549 cells. (h) MS2-RIP followed by miRNA-specific RT-PCR analysis to detect the association of miR-205 with lncRNA-PNUTS in NMuMG cells. LncRNA-PNUTS and GAPDH expression were used as internal controls. (i) A549 and NMUMG cell lysates incubated with in vitro transcribed biotin-labeled lncRNA-PNUTS were subjected to pull-down followed by miRNA extraction and analysis by RT-PCR. (j) A549 cells overexpressing lncRNA-PNUTS were transfected with an increasing concentration of a synthetic miR-205 mimic and the lncRNA expression was assessed by RT-PCR. ZEB-1 and CDH1 were used to monitor the efficiency of miR-205 overexpression on mesenchymal-epithelial transition (MET) process. (k) Time course experiment by using RT-PCR analysis of lncRNA-PNUTS levels upon addition of 10µg.mL −1 cycloheximide in A549 cells. GAPDH was used as a loading control.

    Article Snippet: Sections were incubated overnight in a humid chamber at 4°C with antibody against Ki-67 purchased from Cell signaling technology company (Clone [D2H10], Cat. #9027, 1:1600 dilution) followed by biotinylated secondary antibody (Vector laboratories) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (Dako), and sections were counterstained with hematoxylin before mounting.

    Techniques: Fractionation, Reverse Transcription Polymerase Chain Reaction, Expressing, Confocal Microscopy, Imaging, Cotransfection, Construct, Activation Assay, Quantitative RT-PCR, In Silico, Binding Assay, Incubation, In Vitro, Labeling, Transfection, Concentration Assay, Over Expression