biotinylated rabbit anti goat secondary antibodies  (Vector Laboratories)

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

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

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

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

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:

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

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

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