biotinylated rabbit anti goat secondary antibodies  (Vector Laboratories)


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    Name:
    Biotinylated Rabbit Anti Goat IgG Antibody
    Description:
    Biotinylated Rabbit Anti Goat IgG Antibody is prepared using proprietary immunization schedules that produce high affinity antibodies The antibodies are then purified by affinity chromatography and cross reactivities that are likely to interfere with specific labeling are removed by solid phase adsorption techniques The biotinylated secondary antibodies are conjugated to ensure the maximum degree of labeling without compromising the specificity or affinity of the antibody These antibodies are subjected to rigorous quality control assays and can be used for tissue and cell staining ELISAs and blots Biotinylated Rabbit Anti Goat IgG H L is supplied in liquid format With some exceptions the recommended dilution for most applications is 1 200 H L indicates the antibody recognizes both heavy and light chains The use of bovine products such as casein serum albumin or non fat dry milk as blocking agents in ELISA or blot assays may produce a high background due to cross reactivity of this antibody with bovine immunoglobulins that may be present If this antibody is to be used in these applications blocking with Animal Free Blocker solution Cat No SP 5030 is recommended This antibody is included in the VECTASTAIN ABC kits This antibody is suitable for use with goat sheep and bovine IgG primary antibodies
    Catalog Number:
    BA-5000
    Price:
    None
    Category:
    Antibodies
    Reactivity:
    Goat
    Size:
    1 5 mg
    Host:
    Rabbit
    Buy from Supplier


    Structured Review

    Vector Laboratories biotinylated rabbit anti goat secondary antibodies
    Biotinylated Rabbit Anti Goat IgG Antibody
    Biotinylated Rabbit Anti Goat IgG Antibody is prepared using proprietary immunization schedules that produce high affinity antibodies The antibodies are then purified by affinity chromatography and cross reactivities that are likely to interfere with specific labeling are removed by solid phase adsorption techniques The biotinylated secondary antibodies are conjugated to ensure the maximum degree of labeling without compromising the specificity or affinity of the antibody These antibodies are subjected to rigorous quality control assays and can be used for tissue and cell staining ELISAs and blots Biotinylated Rabbit Anti Goat IgG H L is supplied in liquid format With some exceptions the recommended dilution for most applications is 1 200 H L indicates the antibody recognizes both heavy and light chains The use of bovine products such as casein serum albumin or non fat dry milk as blocking agents in ELISA or blot assays may produce a high background due to cross reactivity of this antibody with bovine immunoglobulins that may be present If this antibody is to be used in these applications blocking with Animal Free Blocker solution Cat No SP 5030 is recommended This antibody is included in the VECTASTAIN ABC kits This antibody is suitable for use with goat sheep and bovine IgG primary antibodies
    https://www.bioz.com/result/biotinylated rabbit anti goat secondary antibodies/product/Vector Laboratories
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated rabbit anti goat secondary antibodies - by Bioz Stars, 2021-04
    95/100 stars

    Images

    1) Product Images from "Expression of orexin A and its receptor 1 in the human prostate"

    Article Title: Expression of orexin A and its receptor 1 in the human prostate

    Journal: Journal of Anatomy

    doi: 10.1111/joa.12030

    OXA- (A and B) and OX1R- (C and D) immunoreactivity in the human normal prostate. (A) Positive cells scattered along the monolayered epithelium of a prostatic follicle. (B) The majority of positive cells visible in this micrograph contain differently stained granular material in the apical portion of their cytoplasm. (C and D) Positive cells lined along the stratified epithelium of three follicles. Some of them are elongated in shape and completely filled with clusters of immunoreactive granules. Avidin–biotin immunohistochemical method. Scale bars: 20 μm.
    Figure Legend Snippet: OXA- (A and B) and OX1R- (C and D) immunoreactivity in the human normal prostate. (A) Positive cells scattered along the monolayered epithelium of a prostatic follicle. (B) The majority of positive cells visible in this micrograph contain differently stained granular material in the apical portion of their cytoplasm. (C and D) Positive cells lined along the stratified epithelium of three follicles. Some of them are elongated in shape and completely filled with clusters of immunoreactive granules. Avidin–biotin immunohistochemical method. Scale bars: 20 μm.

    Techniques Used: Staining, Avidin-Biotin Assay, Immunohistochemistry

    OXA- (A and B) and OX1R- (C and D) immunoreactivity in the human hyperplastic prostate. (A) The basal membrane of an intrafollicular, papillar-like structure is lined by a continuous row of intensely stained cells. (B) A peculiarity often shown by positive basal cells is the presence of a slender cytoplasmic extension (arrows) directed towards the follicular lumen and intermingled between the negative apical cells. (C) Almost all basal cells of the follicular epithelium contain OX1R-immunoreactive material, which completely fills their cytoplasm. (D) Invagination of a prostatic follicle completely lined by positive basal cells. The arrow points to a small cluster of low intensely stained apical cells close in contact with the follicular fluid. Avidin–biotin immunohistochemical method. Scale bars: 20 μm.
    Figure Legend Snippet: OXA- (A and B) and OX1R- (C and D) immunoreactivity in the human hyperplastic prostate. (A) The basal membrane of an intrafollicular, papillar-like structure is lined by a continuous row of intensely stained cells. (B) A peculiarity often shown by positive basal cells is the presence of a slender cytoplasmic extension (arrows) directed towards the follicular lumen and intermingled between the negative apical cells. (C) Almost all basal cells of the follicular epithelium contain OX1R-immunoreactive material, which completely fills their cytoplasm. (D) Invagination of a prostatic follicle completely lined by positive basal cells. The arrow points to a small cluster of low intensely stained apical cells close in contact with the follicular fluid. Avidin–biotin immunohistochemical method. Scale bars: 20 μm.

    Techniques Used: Staining, Avidin-Biotin Assay, Immunohistochemistry

    Related Articles

    Incubation:

    Article Title: Endometrial CXCL13 Expression Is Cycle Regulated in Humans and Aberrantly Expressed in Humans and Rhesus Macaques With Endometriosis
    Article Snippet: Sections were then incubated at 4°C overnight with a goat polyclonal antibody raised to detect CXCL13 (R & D Systems); primary antibody was omitted from slides used as negative controls. .. After overnight incubation with primary antibody, all slides were rinsed in PBS (containing 0.075% nonionic detergent BRIJ), incubated with normal serum for 20 minutes, and then incubated with the biotinylated rabbit anti-goat IgG (Vector Labs) for 30 minutes. .. The slides were rinsed with PBS, reacted with avidin–biotin peroxidase reagents (for 60 minutes), and rinsed in Tris buffer (pH 7.6).

    Article Title: Cholinergic Modulation of Spindle Bursts in the Neonatal Rat Visual Cortex In Vivo
    Article Snippet: Preincubation of the tissue in 10% normal serum (Vector Laboratories, Burlingame, CA) was followed by exposure to the primary antiserum (1:5.000) at 6°C for 36 h under gentle agitation. .. The tissue was afterward incubated with biotinylated rabbit anti-goat serum (1:200; Vector Laboratories) for 2 h at room temperature (RT) and subsequently treated with peroxidase coupled to an avidin-biotin complex (ABC, 1:400; Vector Laboratories) for 2 h at RT. ..

    Article Title: Peroxisome Proliferator-Activated Receptor- γ Is a Potent Target for Prevention and Treatment in Human Prostate and Testicular Cancer
    Article Snippet: PCR products were run on 2% agarose gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA) and visualized by ethidium bromide staining. .. Immunohistochemistry Tissues sections (4 μ m thick) were incubated with anti-PPAR- α , - β , and - γ antibody (2 μ g/mL) or purified normal goat IgG (2 μ g/mL) in a humid chamber for 24 hours, and further incubated with biotinylated rabbit antigoat IgG (Vector Laboratories, Inc. Burlingame, Calif, USA) for 30 minutes. .. After washing with PBS, the sections were incubated with the vectastatin avidin-biotin peroxidase complex kit (Vector, Burlingame, Calif, USA) [ ] for 45 minutes.

    Blocking Assay:

    Article Title: Expression of orexin A and its receptor 1 in the human prostate
    Article Snippet: .. Goat polyclonal anti-OXA (sc-8070) and anti-OX1R antibodies (sc-8073), their respective blocking peptides (sc-8070 P, sc-8073 P), and mouse monoclonal anti-chromogranin A (Chr A) antibody (sc-47714) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-prepro-orexin antibody (AB3096), its blocking peptide (AG774) and monoclonal anti-tubulin antibody (MAB1637) from Chemicon International (Temecula, CA, USA); biotinylated rabbit anti-goat secondary antibodies and avidin–biotin complex (PK-6105) from Vector Laboratories (Burlingame, CA, USA); horseradish peroxidase-conjugated anti-goat IgG (A-5420) and anti-rabbit IgG (A-0545) from Sigma Chemical (St Louis, MO, USA). .. Triazol (15596-026) was purchased from Invitrogen (Carlsbad, CA, USA); the enhanced chemiluminescence (ECL) kit (RPN2209), the GFX PCR DNA and Gel Purification Kit (27-9602-01) from Amersham (Little Chalfont, UK); the DC protein assay kit (500-0111) from Bio-Rad Laboratories (Hercules, CA, USA); the primers for human prepro-orexin, OX1R and β-actin from Primm (Milan, Italy); the kit for PCR and RT-PCR (A1280) from Promega (Madison, WI, USA); and ethylenediamine tetra-acetic-acid (EDTA; 60-00-4) from Sigma Aldrich.

    Avidin-Biotin Assay:

    Article Title: Expression of orexin A and its receptor 1 in the human prostate
    Article Snippet: .. Goat polyclonal anti-OXA (sc-8070) and anti-OX1R antibodies (sc-8073), their respective blocking peptides (sc-8070 P, sc-8073 P), and mouse monoclonal anti-chromogranin A (Chr A) antibody (sc-47714) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-prepro-orexin antibody (AB3096), its blocking peptide (AG774) and monoclonal anti-tubulin antibody (MAB1637) from Chemicon International (Temecula, CA, USA); biotinylated rabbit anti-goat secondary antibodies and avidin–biotin complex (PK-6105) from Vector Laboratories (Burlingame, CA, USA); horseradish peroxidase-conjugated anti-goat IgG (A-5420) and anti-rabbit IgG (A-0545) from Sigma Chemical (St Louis, MO, USA). .. Triazol (15596-026) was purchased from Invitrogen (Carlsbad, CA, USA); the enhanced chemiluminescence (ECL) kit (RPN2209), the GFX PCR DNA and Gel Purification Kit (27-9602-01) from Amersham (Little Chalfont, UK); the DC protein assay kit (500-0111) from Bio-Rad Laboratories (Hercules, CA, USA); the primers for human prepro-orexin, OX1R and β-actin from Primm (Milan, Italy); the kit for PCR and RT-PCR (A1280) from Promega (Madison, WI, USA); and ethylenediamine tetra-acetic-acid (EDTA; 60-00-4) from Sigma Aldrich.

    Article Title: Cholinergic Modulation of Spindle Bursts in the Neonatal Rat Visual Cortex In Vivo
    Article Snippet: Preincubation of the tissue in 10% normal serum (Vector Laboratories, Burlingame, CA) was followed by exposure to the primary antiserum (1:5.000) at 6°C for 36 h under gentle agitation. .. The tissue was afterward incubated with biotinylated rabbit anti-goat serum (1:200; Vector Laboratories) for 2 h at room temperature (RT) and subsequently treated with peroxidase coupled to an avidin-biotin complex (ABC, 1:400; Vector Laboratories) for 2 h at RT. ..

    Amplification:

    Article Title: Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells
    Article Snippet: Primary antibodies used include a goat polyclonal anti-JAG1 (SC-6011; Santa Cruz Biotechnology, Dallas, TX), a rabbit monoclonal anti-P450SCC (14217; Cell Signaling Technology, Danvers, MA), and a rabbit polyclonal anti-GFP ( ; Life Technology, Eugene, OR). .. Biotinylated conjugated secondary antibodies against goat (BA-5000) or rabbit (BA-1000) were purchased from Vector Laboratories (Burlingame, CA) and used with a Colorimetric Tyramide Signal Amplification kit (for GFP IHC; PerkinElmer, Waltham, MA) or a VECTASTAIN Elite ABC Kit (Vector Laboratories). ..

    Immunohistochemistry:

    Article Title: Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells
    Article Snippet: Primary antibodies used include a goat polyclonal anti-JAG1 (SC-6011; Santa Cruz Biotechnology, Dallas, TX), a rabbit monoclonal anti-P450SCC (14217; Cell Signaling Technology, Danvers, MA), and a rabbit polyclonal anti-GFP ( ; Life Technology, Eugene, OR). .. Biotinylated conjugated secondary antibodies against goat (BA-5000) or rabbit (BA-1000) were purchased from Vector Laboratories (Burlingame, CA) and used with a Colorimetric Tyramide Signal Amplification kit (for GFP IHC; PerkinElmer, Waltham, MA) or a VECTASTAIN Elite ABC Kit (Vector Laboratories). ..

    Article Title: Peroxisome Proliferator-Activated Receptor- γ Is a Potent Target for Prevention and Treatment in Human Prostate and Testicular Cancer
    Article Snippet: PCR products were run on 2% agarose gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA) and visualized by ethidium bromide staining. .. Immunohistochemistry Tissues sections (4 μ m thick) were incubated with anti-PPAR- α , - β , and - γ antibody (2 μ g/mL) or purified normal goat IgG (2 μ g/mL) in a humid chamber for 24 hours, and further incubated with biotinylated rabbit antigoat IgG (Vector Laboratories, Inc. Burlingame, Calif, USA) for 30 minutes. .. After washing with PBS, the sections were incubated with the vectastatin avidin-biotin peroxidase complex kit (Vector, Burlingame, Calif, USA) [ ] for 45 minutes.

    Purification:

    Article Title: Peroxisome Proliferator-Activated Receptor- γ Is a Potent Target for Prevention and Treatment in Human Prostate and Testicular Cancer
    Article Snippet: PCR products were run on 2% agarose gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA) and visualized by ethidium bromide staining. .. Immunohistochemistry Tissues sections (4 μ m thick) were incubated with anti-PPAR- α , - β , and - γ antibody (2 μ g/mL) or purified normal goat IgG (2 μ g/mL) in a humid chamber for 24 hours, and further incubated with biotinylated rabbit antigoat IgG (Vector Laboratories, Inc. Burlingame, Calif, USA) for 30 minutes. .. After washing with PBS, the sections were incubated with the vectastatin avidin-biotin peroxidase complex kit (Vector, Burlingame, Calif, USA) [ ] for 45 minutes.

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    Vector Laboratories biotinylated secondary goat anti mouse antibody
    Assessments based on enzyme-linked immunosorbent assay of bound Aβ 1-42 -biotin does not distinguish between elderly high-pathology controls and elderly subjects with mild ADD. ( A ) There was no difference between groups in the levels of overall Aβ 1-42 -biotin recovered from dissociated tissue, as measured using an indirect ELISA which only detects <t>biotinylated</t> Aβ (not significant [n.s.] by Mann–Whitney U test). Data expressed as picograms Aβ per nanogram of total measurable protein. ( B ) The ratio of the amount of Aβ 1-42 -biotin to the amount of Aβ 1-x as measured by sandwich ELISA was not different between groups (not significant [n.s.] by Mann–Whitney U test). ( C ) The ratio of Aβ Aβ 1-42 -biotin as measured by sandwich ELISA to the percent gray matter plaque coverage did not differ between groups (not significant [n.s.] by Mann–Whitney U test). (D) There was no difference between groups in the levels of overall Aβ 1-x recovered from dissociated tissue, as measured using an sandwich ELISA (not significant [n.s.] by Mann–Whitney U test).
    Biotinylated Secondary Goat Anti Mouse Antibody, 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 goat anti mouse antibody/product/Vector Laboratories
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated secondary goat anti mouse antibody - by Bioz Stars, 2021-04
    99/100 stars
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    99
    Vector Laboratories biotinylated goat anti rabbit secondary antisera
    IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with <t>biotinylated</t> secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p
    Biotinylated Goat Anti Rabbit Secondary Antisera, 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 goat anti rabbit secondary antisera/product/Vector Laboratories
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    biotinylated goat anti rabbit secondary antisera - by Bioz Stars, 2021-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Assessments based on enzyme-linked immunosorbent assay of bound Aβ 1-42 -biotin does not distinguish between elderly high-pathology controls and elderly subjects with mild ADD. ( A ) There was no difference between groups in the levels of overall Aβ 1-42 -biotin recovered from dissociated tissue, as measured using an indirect ELISA which only detects biotinylated Aβ (not significant [n.s.] by Mann–Whitney U test). Data expressed as picograms Aβ per nanogram of total measurable protein. ( B ) The ratio of the amount of Aβ 1-42 -biotin to the amount of Aβ 1-x as measured by sandwich ELISA was not different between groups (not significant [n.s.] by Mann–Whitney U test). ( C ) The ratio of Aβ Aβ 1-42 -biotin as measured by sandwich ELISA to the percent gray matter plaque coverage did not differ between groups (not significant [n.s.] by Mann–Whitney U test). (D) There was no difference between groups in the levels of overall Aβ 1-x recovered from dissociated tissue, as measured using an sandwich ELISA (not significant [n.s.] by Mann–Whitney U test).

    Journal: PLoS ONE

    Article Title: Soluble amyloid-beta buffering by plaques in Alzheimer disease dementia versus high-pathology controls

    doi: 10.1371/journal.pone.0200251

    Figure Lengend Snippet: Assessments based on enzyme-linked immunosorbent assay of bound Aβ 1-42 -biotin does not distinguish between elderly high-pathology controls and elderly subjects with mild ADD. ( A ) There was no difference between groups in the levels of overall Aβ 1-42 -biotin recovered from dissociated tissue, as measured using an indirect ELISA which only detects biotinylated Aβ (not significant [n.s.] by Mann–Whitney U test). Data expressed as picograms Aβ per nanogram of total measurable protein. ( B ) The ratio of the amount of Aβ 1-42 -biotin to the amount of Aβ 1-x as measured by sandwich ELISA was not different between groups (not significant [n.s.] by Mann–Whitney U test). ( C ) The ratio of Aβ Aβ 1-42 -biotin as measured by sandwich ELISA to the percent gray matter plaque coverage did not differ between groups (not significant [n.s.] by Mann–Whitney U test). (D) There was no difference between groups in the levels of overall Aβ 1-x recovered from dissociated tissue, as measured using an sandwich ELISA (not significant [n.s.] by Mann–Whitney U test).

    Article Snippet: The following day, sections were washed 3x in TBS for 5 minutes each and incubated with a biotinylated secondary goat anti-mouse antibody at a 1:1000 dilution in TBS-X for 1 hour at room temperature (Vector Laboratories).

    Techniques: Enzyme-linked Immunosorbent Assay, Indirect ELISA, MANN-WHITNEY, Sandwich ELISA

    IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p

    Journal: eNeuro

    Article Title: Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network

    doi: 10.1523/ENEURO.0400-18.2019

    Figure Lengend Snippet: IHC for POMC cell counts in control and restored mice, and from VGlut2-Cre; tdTomato animals. A , POMC-IR in a male control mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). B , POMC-IR in a male restored mouse detected with an Alexa Fluor 568 (red) secondary antibody (1:500). C , POMC-IR in a female control mouse detected with biotinylated secondary antibody (1:500) and visualized with a diaminobenzidine (DAB) reaction (brown). D , POMC-IR in a female restored mouse detected with biotinylated secondary antibody (1:500) and visualized with a DAB reaction (brown). E , POMC-IR in a female Vglut2-Cre; tdTomato mouse detected with an Alexa Fluor 488 (green) secondary antibody (1:500; mirrored section from Fig. 1 G , H ). F , POMC neuron cell counts from sections (three per mouse). There was no difference between control (blue bars) or restored (green bars) mice, but only in the method of secondary labeling used. Male data for each group represented by filled blue circles and female data shown by filled pink circles; **** p

    Article Snippet: Following triplicate washes, one set of the sections from control and restored mice was incubated with a biotinylated goat anti-rabbit secondary antisera (1:500, Vector Labs; catalog #BA-1000 ROS23) followed by treatment with a Vectastain ABC HRP kit (Vector Labs; catalog #PK-4000) and development of a colorimetric stain with diaminobenzidine (250 μg/ml in TBS with 0.1% H2 O2 ).

    Techniques: Immunohistochemistry, Mouse Assay, Labeling

    Characterization of total and phosphorylated S129 human alpha-synuclein duplex assay . The hook-point for biotinylated 4B12 antibody was determined simultaneously in a mix of Europium Acceptor-bead coupled 11A5 antibody and Terbium Acceptor-bead coupled LB509 antibody ( a ). Arrows indicate optimal 4B12 concentrations for both Acceptor-bead variants. Equivalency of the signal obtained in the total h-asyn assay was demonstrated for pS129 h-asyn and h-asyn proteins in the duplex assay in two consecutive experiments and data plotted in panel ( b ). All protein standards were spiked in naïve rat brain lysate. Lower detection limit (LDL) was 450 pg/ml and is indicated with a dashed line. Intra assay variation was calculated based on three standard curves which were measured on the same day but on separate plates for each emission signal separately ( c and d ). Inter assay variation data was calculated based on six pairs of standard curves performed on separate days ( e and f ). AU arbitrary units

    Journal: Molecular Neurodegeneration

    Article Title: A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

    doi: 10.1186/s13024-016-0125-0

    Figure Lengend Snippet: Characterization of total and phosphorylated S129 human alpha-synuclein duplex assay . The hook-point for biotinylated 4B12 antibody was determined simultaneously in a mix of Europium Acceptor-bead coupled 11A5 antibody and Terbium Acceptor-bead coupled LB509 antibody ( a ). Arrows indicate optimal 4B12 concentrations for both Acceptor-bead variants. Equivalency of the signal obtained in the total h-asyn assay was demonstrated for pS129 h-asyn and h-asyn proteins in the duplex assay in two consecutive experiments and data plotted in panel ( b ). All protein standards were spiked in naïve rat brain lysate. Lower detection limit (LDL) was 450 pg/ml and is indicated with a dashed line. Intra assay variation was calculated based on three standard curves which were measured on the same day but on separate plates for each emission signal separately ( c and d ). Inter assay variation data was calculated based on six pairs of standard curves performed on separate days ( e and f ). AU arbitrary units

    Article Snippet: The next day sections were rinsed with KPBS and incubated in 0.25 % Triton + 5 % normal serum in KPBS for 1 h containing secondary antibody biotinylated goat anti rabbit (1:200, Vector Laboratories Inc., USA).

    Techniques: Intra Assay, Inter Assay

    Characterization of total human alpha-synuclein AlphaLISA. The hook-point analysis was evaluated for serial dilutions of biotinylated 4B12 in combination with Europium Acceptor-beads coupled to either LB509 (LB509-Eu) or syn211 (syn211-Eu) using a 19 ng/ml of recombinant GST-tagged h-asyn protein diluted in 10 μg/ml of Tris wild-type rat brain lysate ( a ). Arrow indicates the highest signal obtained and therefore optimal 4B12-biotin concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled LB509 by serial dilution of either GST-tagged h-asyn or mouse asyn (m-asyn) protein spiked in Tris brain lysate obtained from a naïve rat (10 μg/ml) ( b ). The lower detection limit (LDL) was 3.7 pg/ml and is indicated by a dashed line . Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ). Inter assay variation data was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units

    Journal: Molecular Neurodegeneration

    Article Title: A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

    doi: 10.1186/s13024-016-0125-0

    Figure Lengend Snippet: Characterization of total human alpha-synuclein AlphaLISA. The hook-point analysis was evaluated for serial dilutions of biotinylated 4B12 in combination with Europium Acceptor-beads coupled to either LB509 (LB509-Eu) or syn211 (syn211-Eu) using a 19 ng/ml of recombinant GST-tagged h-asyn protein diluted in 10 μg/ml of Tris wild-type rat brain lysate ( a ). Arrow indicates the highest signal obtained and therefore optimal 4B12-biotin concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled LB509 by serial dilution of either GST-tagged h-asyn or mouse asyn (m-asyn) protein spiked in Tris brain lysate obtained from a naïve rat (10 μg/ml) ( b ). The lower detection limit (LDL) was 3.7 pg/ml and is indicated by a dashed line . Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ). Inter assay variation data was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units

    Article Snippet: The next day sections were rinsed with KPBS and incubated in 0.25 % Triton + 5 % normal serum in KPBS for 1 h containing secondary antibody biotinylated goat anti rabbit (1:200, Vector Laboratories Inc., USA).

    Techniques: Recombinant, Concentration Assay, Serial Dilution, Intra Assay, Inter Assay

    Evaluation of the duplex AlphaLISA performance. Assessment of the Acceptor-bead performance and comparison between Europium and Terbium based beads was carried out once for both the LB509 ( a ) and the 11A5 ( b ) antibodies. In both instances the biotinylated 4B12 antibody was used and signal to background ratio calculated against serial dilutions of h-asyn proteins spiked in naïve rat brain lysate. Presence of any hindrance of Acceptor-bead coupled antibodies against each other was assessed for LB509-Terbium ( c ) and 11A5-Europium beads ( d ). Cross-talk between the channels was assessed using the Resorufine/Amplex Red FP535 Terbium filter ( e ) and the Europium 615 filter ( f ). For these experiments the relevant analyte, Acceptor- and Donor-bead information is given under the x-axis. Both the hindrance of Acceptor-bead coupled antibodies and the channel cross talk were run twice. See Additional file 2 : Figure S2 for a theoretical explanation of the phenomena. AU arbitrary units

    Journal: Molecular Neurodegeneration

    Article Title: A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

    doi: 10.1186/s13024-016-0125-0

    Figure Lengend Snippet: Evaluation of the duplex AlphaLISA performance. Assessment of the Acceptor-bead performance and comparison between Europium and Terbium based beads was carried out once for both the LB509 ( a ) and the 11A5 ( b ) antibodies. In both instances the biotinylated 4B12 antibody was used and signal to background ratio calculated against serial dilutions of h-asyn proteins spiked in naïve rat brain lysate. Presence of any hindrance of Acceptor-bead coupled antibodies against each other was assessed for LB509-Terbium ( c ) and 11A5-Europium beads ( d ). Cross-talk between the channels was assessed using the Resorufine/Amplex Red FP535 Terbium filter ( e ) and the Europium 615 filter ( f ). For these experiments the relevant analyte, Acceptor- and Donor-bead information is given under the x-axis. Both the hindrance of Acceptor-bead coupled antibodies and the channel cross talk were run twice. See Additional file 2 : Figure S2 for a theoretical explanation of the phenomena. AU arbitrary units

    Article Snippet: The next day sections were rinsed with KPBS and incubated in 0.25 % Triton + 5 % normal serum in KPBS for 1 h containing secondary antibody biotinylated goat anti rabbit (1:200, Vector Laboratories Inc., USA).

    Techniques:

    Characterization of S129 phosphorylated human alpha-synuclein specific AlphaLISA. The hook-point analysis was evaluated for biotinylated 4B12 and Europium Acceptor-bead conjugated 11A5 ( a ). Pair was tested using 14 ng/ml of pS129 h-asyn protein diluted in 10 μg/ml of Tris brain lysate obtained from an asyn knockout mouse. Arrow indicates the optimal biotinylated antibody concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled 11A5 by serial dilution of either pS129 h-asyn or S129A h-asyn protein spiked in wild-type rodent brain lysate (10 μg/ml) ( b ). The lower detection limit (LDL) was 1.1 pg/ml and is indicated by a dashed line. Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ) while the inter assay variation was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units

    Journal: Molecular Neurodegeneration

    Article Title: A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

    doi: 10.1186/s13024-016-0125-0

    Figure Lengend Snippet: Characterization of S129 phosphorylated human alpha-synuclein specific AlphaLISA. The hook-point analysis was evaluated for biotinylated 4B12 and Europium Acceptor-bead conjugated 11A5 ( a ). Pair was tested using 14 ng/ml of pS129 h-asyn protein diluted in 10 μg/ml of Tris brain lysate obtained from an asyn knockout mouse. Arrow indicates the optimal biotinylated antibody concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled 11A5 by serial dilution of either pS129 h-asyn or S129A h-asyn protein spiked in wild-type rodent brain lysate (10 μg/ml) ( b ). The lower detection limit (LDL) was 1.1 pg/ml and is indicated by a dashed line. Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates ( c ) while the inter assay variation was calculated based on two pairs of standard curves performed on separate days ( d ). AU arbitrary units

    Article Snippet: The next day sections were rinsed with KPBS and incubated in 0.25 % Triton + 5 % normal serum in KPBS for 1 h containing secondary antibody biotinylated goat anti rabbit (1:200, Vector Laboratories Inc., USA).

    Techniques: Knock-Out, Concentration Assay, Serial Dilution, Intra Assay, Inter Assay

    PEDF-deficiency protects against IL-1β-induced MMP-1, MMP-3 and MMP-13 protein production in metatarsal bone cultures. Metatarsal bones from 10 and 29 week old wild type or PEDF-deficient mice were harvested and cultured in the presence or absence of 10 ng/mL IL-1β for 7 days. Cryosectioned samples were analyzed for MMP protein levels by immunohistochemistry (IHC). AC: articular cartilage. SB: subchondral bone. Scale bar = 100 μm. a Samples were stained with rabbit anti-MMP-1 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-1 staining. b Samples were stained with rabbit anti-MMP-3 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-3 staining. c Samples were stained with mouse anti-MMP-13 and nuclei were stained with DAPI. Arrows indicate positive MMP-13 staining. The percent area staining positive for MMP-13 in the 10 and 29 week old samples was calculated as the area with MMP-13 staining along the articular cartilage area against the total articular cartilage area. For experiments involving either 10 or 29 week old mice, the middle three metatarsal bones isolated from three animals were used. Each data point in the graphs represents an individual metatarsal bone. Each treatment was repeated 5–11 times. Negative controls using goat anti-rabbit IgG or biotinylated horse anti-mouse IgG followed by streptavidin DyLight 594 alone are shown in the Additional file 2 Figure S1. Data are plotted as mean ± SEM. * p = 0.0281 (Mann–Whitney test)

    Journal: BMC Musculoskeletal Disorders

    Article Title: Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions

    doi: 10.1186/s12891-017-1410-y

    Figure Lengend Snippet: PEDF-deficiency protects against IL-1β-induced MMP-1, MMP-3 and MMP-13 protein production in metatarsal bone cultures. Metatarsal bones from 10 and 29 week old wild type or PEDF-deficient mice were harvested and cultured in the presence or absence of 10 ng/mL IL-1β for 7 days. Cryosectioned samples were analyzed for MMP protein levels by immunohistochemistry (IHC). AC: articular cartilage. SB: subchondral bone. Scale bar = 100 μm. a Samples were stained with rabbit anti-MMP-1 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-1 staining. b Samples were stained with rabbit anti-MMP-3 and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate. Arrows indicate positive MMP-3 staining. c Samples were stained with mouse anti-MMP-13 and nuclei were stained with DAPI. Arrows indicate positive MMP-13 staining. The percent area staining positive for MMP-13 in the 10 and 29 week old samples was calculated as the area with MMP-13 staining along the articular cartilage area against the total articular cartilage area. For experiments involving either 10 or 29 week old mice, the middle three metatarsal bones isolated from three animals were used. Each data point in the graphs represents an individual metatarsal bone. Each treatment was repeated 5–11 times. Negative controls using goat anti-rabbit IgG or biotinylated horse anti-mouse IgG followed by streptavidin DyLight 594 alone are shown in the Additional file 2 Figure S1. Data are plotted as mean ± SEM. * p = 0.0281 (Mann–Whitney test)

    Article Snippet: For colorimetric staining, a biotinylated anti-mouse IgG secondary antibody (Vector Laboratories) or a goat anti-rabbit IgG secondary antibody (Vector Laboratories) was used, followed by the Vectastain ABC Kit with DAB Peroxidase Substrate Kit (Vector Laboratories), and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate.

    Techniques: Mouse Assay, Cell Culture, Immunohistochemistry, Staining, Isolation, MANN-WHITNEY

    PEDF expression is elevated in human osteoarthritis (OA) cartilage specimens. Normal samples were obtained from the National Disease Research Interchange and OA samples were obtained from patients undergoing total knee replacement surgery. a Normal sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 1. b Immunohistochemistry (IHC) analysis on a normal cartilage sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (b') and deeper (b'') zones are shown from insets. c OA sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 5. d IHC analysis on an OA sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (d') and deeper (d'') zones are shown from insets. Arrows indicate positive PEDF staining. A negative control using a biotinylated horse anti-mouse IgG secondary antibody alone is shown in the Additional file 2 : Figure S1. Scale bar = 200 μm

    Journal: BMC Musculoskeletal Disorders

    Article Title: Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions

    doi: 10.1186/s12891-017-1410-y

    Figure Lengend Snippet: PEDF expression is elevated in human osteoarthritis (OA) cartilage specimens. Normal samples were obtained from the National Disease Research Interchange and OA samples were obtained from patients undergoing total knee replacement surgery. a Normal sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 1. b Immunohistochemistry (IHC) analysis on a normal cartilage sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (b') and deeper (b'') zones are shown from insets. c OA sample stained with Safranin O and counterstained with Hematoxylin and Fast Green. Mankin score = 5. d IHC analysis on an OA sample using a mouse anti-PEDF antibody and counterstained with Methyl Green. Magnified superficial (d') and deeper (d'') zones are shown from insets. Arrows indicate positive PEDF staining. A negative control using a biotinylated horse anti-mouse IgG secondary antibody alone is shown in the Additional file 2 : Figure S1. Scale bar = 200 μm

    Article Snippet: For colorimetric staining, a biotinylated anti-mouse IgG secondary antibody (Vector Laboratories) or a goat anti-rabbit IgG secondary antibody (Vector Laboratories) was used, followed by the Vectastain ABC Kit with DAB Peroxidase Substrate Kit (Vector Laboratories), and counterstained with 0.5% Methyl Green in 0.1 M Sodium Acetate.

    Techniques: Expressing, Staining, Immunohistochemistry, Negative Control