peroxidase streptavidin  (Thermo Fisher)


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    Structured Review

    Thermo Fisher peroxidase streptavidin
    Peroxidase Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/peroxidase streptavidin/product/Thermo Fisher
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    peroxidase streptavidin - by Bioz Stars, 2020-03
    99/100 stars

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    Related Articles

    Histopathology:

    Article Title: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape
    Article Snippet: Paragraph title: Histopathology ... Treg cells were detected using a rat anti-mouse FoxP3 antibody (14-5773-82, eBioscience, San Diego, CA) with heat induced retrieval, pH 6.0 Target Retrieval Buffer (S699, DAKO, Carpinteria, CA) followed by a horseradish peroxidase labeled streptavidin detection system (TS-125-HR, Thermo Shandon, Pittsburgh, PA).

    Immunohistochemistry:

    Article Title: Ganoderma lucidum, a promising agent possessing antioxidant and anti-inflammatory effects for treating calvarial defects with graft application in rats 1
    Article Snippet: Paragraph title: Immunohistochemical staining ... The sections were washed 3 times for 5 min in PBS and then were incubated with biotinylated secondary antibody (catalogue #TP-125-BN, Thermo Fisher Scientific, US) for 14 min. After washing with PBS, streptavidin peroxidase (catalogue #TS-125-HR, Thermo Fisher Scientific, US) was applied to the sections for 15 min.

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: Paragraph title: Immunohistochemistry ... After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape
    Article Snippet: Treg cells were detected using a rat anti-mouse FoxP3 antibody (14-5773-82, eBioscience, San Diego, CA) with heat induced retrieval, pH 6.0 Target Retrieval Buffer (S699, DAKO, Carpinteria, CA) followed by a horseradish peroxidase labeled streptavidin detection system (TS-125-HR, Thermo Shandon, Pittsburgh, PA). .. In all immunohistochemical assays, 3,3′-diaminobenzidine was used as the chromogenic substrate with a light hematoxylin counterstain.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: Paragraph title: Immunohistochemistry ... Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin.

    Article Title: Immunoexpression of cyclooxygenase-2 in primary gastric carcinomas and lymph node metastases
    Article Snippet: Paragraph title: Immunohistochemistry ... A rabbit monoclonal antibody against human COX-2 (Clone SP21, pm 70 kDa, LabVision Co™, 1:200) was applied for 60 min. After washing with phosphate buffered saline (PBS), the sections were incubated with a secondary anti-polyvalent biotinylated goat antibody (TP-125-BN, LabVision Co™) for 15 min, washed with PBS, incubated with a streptavidin-coupled peroxidase complex (TS-125-HR, LabVision Co™) for 15 min, then washed again with PBS.

    Article Title: Primary Epiphyseal Arteriopathy in a Mouse Model of Steroid-Induced Osteonecrosis
    Article Snippet: Paragraph title: Immunohistochemistry ... Bound antibodies were detected with the horseradish peroxidase-labeled streptavidin method (catalog #TS-125-HR; ThermoShandon), with 3,3′-diaminobenzidine (catalog #TA-125-HDX; ThermoShandon) as chromogenic substrate (DAB; catalog #TA-125-HDX; ThermoShandon).

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: Paragraph title: Immunohistochemical evaluation ... After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature.

    Article Title: Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH)
    Article Snippet: Paragraph title: Analysis of vascular density and bone formation by immunohistochemistry ... After being washed with PBS, the sections were treated with streptavidin peroxidase (TS-125-HR, Labvision, Fremont, CA, USA) and then with 3,3’-diaminobenzidine (DAB) chromogen for 30 seconds.

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: Paragraph title: Immunohistochemistry ... Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody.

    Article Title: Enhanced Susceptibility of Ago1/3 Double-Null Mice to Influenza A Virus Infection
    Article Snippet: Paragraph title: Lung pathological analysis and influenza virus immunohistochemistry. ... Detection of bound secondary antibodies was performed with horseradish peroxidase-labeled streptavidin (catalog number TS-125-HR; Thermo Scientific, Kalamazoo, MI) with 3,3′ diaminobenzidine (DAB; 5 min; catalog number TA-125-HDX; Thermo Shandon) as chromogenic substrate and a light hematoxylin counterstain (catalog number TA-125-MH; Thermo Scientific, Kalamazoo, MI).

    Article Title: ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma
    Article Snippet: Paragraph title: Immunohistochemistry (IHC) ... Next, slides were washed and incubated in streptavidin solution (Thermoscientific cat. no. TS-060-HR).

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: Paragraph title: 2.1. Immunohistochemistry ... The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA).

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: Paragraph title: Immunohistochemistry ... After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS.

    Injection:

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: .. Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody. .. After blocking, the section was incubated overnight with 100-fold diluted primary antibody and rinsed with PBS.

    Positive Control:

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin. .. Serial sections of canine lymph node were used as positive control.

    Labeling:

    Article Title: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape
    Article Snippet: .. Treg cells were detected using a rat anti-mouse FoxP3 antibody (14-5773-82, eBioscience, San Diego, CA) with heat induced retrieval, pH 6.0 Target Retrieval Buffer (S699, DAKO, Carpinteria, CA) followed by a horseradish peroxidase labeled streptavidin detection system (TS-125-HR, Thermo Shandon, Pittsburgh, PA). .. In all immunohistochemical assays, 3,3′-diaminobenzidine was used as the chromogenic substrate with a light hematoxylin counterstain.

    Article Title: Primary Epiphyseal Arteriopathy in a Mouse Model of Steroid-Induced Osteonecrosis
    Article Snippet: For the labeling of α-SMA, the primary antibody was first biotinylated with the ARK kit (catalog #K3954; Dako) according to the manufacturer’s instructions. .. Bound antibodies were detected with the horseradish peroxidase-labeled streptavidin method (catalog #TS-125-HR; ThermoShandon), with 3,3′-diaminobenzidine (catalog #TA-125-HDX; ThermoShandon) as chromogenic substrate (DAB; catalog #TA-125-HDX; ThermoShandon).

    Plasmid Preparation:

    Article Title: Primary Epiphyseal Arteriopathy in a Mouse Model of Steroid-Induced Osteonecrosis
    Article Snippet: Sections were incubated with anti-CD31 antibody at 1:20 for 60 minutes and then with the secondary biotinylated rabbit anti-rat antibody (Vector; catalog #BA-4001; Burlingame, CA) at 1:200 for 10 minutes. .. Bound antibodies were detected with the horseradish peroxidase-labeled streptavidin method (catalog #TS-125-HR; ThermoShandon), with 3,3′-diaminobenzidine (catalog #TA-125-HDX; ThermoShandon) as chromogenic substrate (DAB; catalog #TA-125-HDX; ThermoShandon).

    Avidin-Biotin Assay:

    Article Title: Enhanced Susceptibility of Ago1/3 Double-Null Mice to Influenza A Virus Infection
    Article Snippet: Endogenous avidin and biotin (catalog number X0590; Dako, Carpinteria, CA) were blocked (10 min each), followed by blocking for 30 min in Superblock (catalog number 37545ZZ; Thermo Scientific Pierce, Rockford, IL). .. Detection of bound secondary antibodies was performed with horseradish peroxidase-labeled streptavidin (catalog number TS-125-HR; Thermo Scientific, Kalamazoo, MI) with 3,3′ diaminobenzidine (DAB; 5 min; catalog number TA-125-HDX; Thermo Shandon) as chromogenic substrate and a light hematoxylin counterstain (catalog number TA-125-MH; Thermo Scientific, Kalamazoo, MI).

    Knock-Out:

    Article Title: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape
    Article Snippet: Tissues from knockout and control mice were fixed in 10% neutral buffered formalin, except eyes, which were fixed for 24 h in Davidson’s fixative, embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin for histopathologic examination. .. Treg cells were detected using a rat anti-mouse FoxP3 antibody (14-5773-82, eBioscience, San Diego, CA) with heat induced retrieval, pH 6.0 Target Retrieval Buffer (S699, DAKO, Carpinteria, CA) followed by a horseradish peroxidase labeled streptavidin detection system (TS-125-HR, Thermo Shandon, Pittsburgh, PA).

    Mouse Assay:

    Article Title: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape
    Article Snippet: Tissues from knockout and control mice were fixed in 10% neutral buffered formalin, except eyes, which were fixed for 24 h in Davidson’s fixative, embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin for histopathologic examination. .. Treg cells were detected using a rat anti-mouse FoxP3 antibody (14-5773-82, eBioscience, San Diego, CA) with heat induced retrieval, pH 6.0 Target Retrieval Buffer (S699, DAKO, Carpinteria, CA) followed by a horseradish peroxidase labeled streptavidin detection system (TS-125-HR, Thermo Shandon, Pittsburgh, PA).

    Light Microscopy:

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber. .. Both the intensity and distribution of iNOS, IL-6, L-FABP, NGAL staining were scored in 10 different areas for each samples under light microscope at magnification X400 in the kidney slices.

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature. .. Then, 3 amino 9-ethylcarbazol (AEC) solution (Large Volume AEC Substrate System (RTU), Thermo Fisher Scientific, TA-060- HA, Fremont, California, USA) was dropped into the tissues and the tissues were examined by light microscopy.

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA). .. After the solution of 3-amino-9-ethylcarbazole (AEC) Substrate+AEC Chromogen (AEC Substrate, TA-015 and HAS; AEC Chromogen, TA-002-HAC, Lab Vision Corporation, USA) was added to the tissues and images taken on a light microscope, it was washed with PBS concurrently.

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS. .. After taking the image signal on a light microscope by dripping the solution of 3-amino-9-ethylcarbazole (AEC) Substrate + AEC Chromogen (AEC Substrate, TA-015 and HAS, AEC Chromogen, TA-002-HAC, LabVision Corporation, USA) on tissues, the tissues were washed with PBS synchronously.

    Incubation:

    Article Title: Ganoderma lucidum, a promising agent possessing antioxidant and anti-inflammatory effects for treating calvarial defects with graft application in rats 1
    Article Snippet: .. The sections were washed 3 times for 5 min in PBS and then were incubated with biotinylated secondary antibody (catalogue #TP-125-BN, Thermo Fisher Scientific, US) for 14 min. After washing with PBS, streptavidin peroxidase (catalogue #TS-125-HR, Thermo Fisher Scientific, US) was applied to the sections for 15 min. ..

    Article Title: The Spermatogonial Stem Cell Niche in the Collared Peccary (Tayassu tajacu) 1
    Article Snippet: .. The strips were washed three times with TBS-0.05% Tween solution, 5 min each, and incubated in streptavidin solution (TS-125-HR; Thermo Scientific) for 15 min, at room temperature. .. These strips were then washed three times with TBS-0.05% Tween solution, 5 min each, and incubated with 3,3′-diaminobenzidine (DAB; Sigma-Aldrich), chloramphenicol (Sigma-Aldrich), and hydrogen peroxide (Sigma-Aldrich) for 1 min, at room temperature, and washed in water.

    Article Title: Spermatogonial Stem Cell Markers and Niche in Equids
    Article Snippet: Subsequently, the strips were rinsed three times with TBS 0.05% Triton-X buffer and then incubated for 1 hour with biotinylated anti-goat (for GFRA1; 1∶100, ab6740, Abcam), anti-mouse (for PLZF; 1∶500, SAB4600004, Sigma) or anti-rabbit (for CSF1R; 1∶200, ab6720, Abcam) IgG antibodies at room temperature. .. Before exposing the strips to streptavidin solution (Thermo Scientific, TS-125-HR) for 15 minutes at room temperature, they were rinsed three times with TBS 0.05% Triton-X buffer.

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: The sections were washed in PBS three times for 5 min each time and then incubated for 30 min at room temperature with biotinylated goat anti-rabbit antibodies (LabVision, TP-125-BN). .. After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: A panel of primary antibodies was applied to serial sections and incubated overnight at 4°C ( ). .. Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin.

    Article Title: Immunoexpression of cyclooxygenase-2 in primary gastric carcinomas and lymph node metastases
    Article Snippet: .. A rabbit monoclonal antibody against human COX-2 (Clone SP21, pm 70 kDa, LabVision Co™, 1:200) was applied for 60 min. After washing with phosphate buffered saline (PBS), the sections were incubated with a secondary anti-polyvalent biotinylated goat antibody (TP-125-BN, LabVision Co™) for 15 min, washed with PBS, incubated with a streptavidin-coupled peroxidase complex (TS-125-HR, LabVision Co™) for 15 min, then washed again with PBS. .. Reactions were processed at room temperature (approximately 20 °C) in an automated immunostainer (Autostainer, LabVision Co™, model 480-2D) at the Institute of Molecular Pathology and Immunology of the University of Porto.

    Article Title: Primary Epiphyseal Arteriopathy in a Mouse Model of Steroid-Induced Osteonecrosis
    Article Snippet: Sections were incubated with the biotinylated primary antibody for 30 minutes. .. Bound antibodies were detected with the horseradish peroxidase-labeled streptavidin method (catalog #TS-125-HR; ThermoShandon), with 3,3′-diaminobenzidine (catalog #TA-125-HDX; ThermoShandon) as chromogenic substrate (DAB; catalog #TA-125-HDX; ThermoShandon).

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature. .. Then, 3 amino 9-ethylcarbazol (AEC) solution (Large Volume AEC Substrate System (RTU), Thermo Fisher Scientific, TA-060- HA, Fremont, California, USA) was dropped into the tissues and the tissues were examined by light microscopy.

    Article Title: Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH)
    Article Snippet: After being blocked with Ultra V Block (Thermo Fisher Scientific, Fremont, CA, USA), the slides were incubated with OPN rabbit polyclonal (1:50; cat.no: RB-9097-P0 Labvision, Thermo Fisher Scientific Fremont, CA, USA) and with CD31 rabbit polyclonal (1:100; bs-0468R Woburn, Massachusetts, USA) antibody dilution for 60 minutes at room temperature. .. After being washed with PBS, the sections were treated with streptavidin peroxidase (TS-125-HR, Labvision, Fremont, CA, USA) and then with 3,3’-diaminobenzidine (DAB) chromogen for 30 seconds.

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody. .. After blocking, the section was incubated overnight with 100-fold diluted primary antibody and rinsed with PBS.

    Article Title: ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma
    Article Snippet: .. Next, slides were washed and incubated in streptavidin solution (Thermoscientific cat. no. TS-060-HR). .. This was followed by incubation in AEC and a rinse in H2 O.

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: .. The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA). .. After the solution of 3-amino-9-ethylcarbazole (AEC) Substrate+AEC Chromogen (AEC Substrate, TA-015 and HAS; AEC Chromogen, TA-002-HAC, Lab Vision Corporation, USA) was added to the tissues and images taken on a light microscope, it was washed with PBS concurrently.

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: .. After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS. .. After taking the image signal on a light microscope by dripping the solution of 3-amino-9-ethylcarbazole (AEC) Substrate + AEC Chromogen (AEC Substrate, TA-015 and HAS, AEC Chromogen, TA-002-HAC, LabVision Corporation, USA) on tissues, the tissues were washed with PBS synchronously.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: Immunohistochemistry Formalin-fixed, paraffin-embedded tissue samples from the surgically resected U87 tumor were cut into 4-mm sections. .. Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody.

    Activity Assay:

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: The endogenous peroxidase activity was blocked with 3% H2 O2 for 15 min at room temperature and later rinsed with distilled water and PBS. .. After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: Immunohistochemistry Sections were dewaxed in xylene and rehydrated through graded alcohols prior to quenching endogenous peroxidase activity with 3% H2 O2 in distilled water for 20 minutes. .. Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin.

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: After washing with PBS for 3×5 minutes, the tissues were incubated in hydrogen peroxide (Hydrogen Peroxide Block, Thermo Fisher Scientific, TA-125-HP, Fremont, California, USA) for 5 minutes to obstruct endogenous peroxidase activity and then washed with PBS for 3×5 minutes. .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature.

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: After boiling, the tissues were incubated for 5 min with hydrogen peroxide block solution (Hydrogen Peroxide Block, TA-125-HP, Lab Vision Corporation, USA) to prevent endogenous peroxidase activity after washing for 3 × 5 min with PBS (Phosphate-Buffered Saline, P4417, Sigma-Aldrich, USA). .. The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA).

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: After boiling, the tissues that were kept to cool off in room heat for about 20 min were incubated for 5 min with hydrogen peroxide block solution to prevent endogenous peroxidase activity after being washed for 3 × 5 min with PBS (Phosphate Buffered Saline, P4417, Sigma-Aldrich, USA) -125-HP, Lab Vision Corporation, USA). .. After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS.

    Blocking Assay:

    Article Title: Ganoderma lucidum, a promising agent possessing antioxidant and anti-inflammatory effects for treating calvarial defects with graft application in rats 1
    Article Snippet: The sections were washed 3 times for 5 min in PBS and incubated with hydrogen peroxide (catalogue #TA-015-HP, Thermo Fisher Scientific, US) for 20 min. Ultra V block (TA-125-UB, Thermo Fisher Scientific, US) was applied to the sections for 8 min prior to the addition of the primary antibodies, which were left on overnight (osteonectin; SPARC monoclonal antibody, catalogue # 33-5500, 1:100; osteopontin monoclonal antibody, catalogue #MA5-17180, 1:100; and MMP-9 monoclonal antibody, catalogue #MA5-13595, 1:200, all from Thermo Fisher Scientific, US). .. The sections were washed 3 times for 5 min in PBS and then were incubated with biotinylated secondary antibody (catalogue #TP-125-BN, Thermo Fisher Scientific, US) for 14 min. After washing with PBS, streptavidin peroxidase (catalogue #TS-125-HR, Thermo Fisher Scientific, US) was applied to the sections for 15 min.

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: Blocking reagent (Lab Vision, TA-125-UB) was applied to each slide followed by 5 min incubation at room temperature in a humid chamber. .. After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: Non-specific antigen binding was blocked by incubation with UltraVision Protein Block (TA-125- PBQ; Thermo Scientific, Cheshire, UK). .. Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin.

    Article Title: Immunoexpression of cyclooxygenase-2 in primary gastric carcinomas and lymph node metastases
    Article Snippet: Endogenous peroxide was blocked with a 3% H2 O2 solution in methanol for 10 min, and for unmasking antigens, a retrieval solution (Vector Co™) 1% in hot water (98 °C) was added and incubated for 20 min. Ultra V block (TA-125-UB, LabVision Co™) was added to the sample and incubated for 10 min to inhibit nonspecific background staining. .. A rabbit monoclonal antibody against human COX-2 (Clone SP21, pm 70 kDa, LabVision Co™, 1:200) was applied for 60 min. After washing with phosphate buffered saline (PBS), the sections were incubated with a secondary anti-polyvalent biotinylated goat antibody (TP-125-BN, LabVision Co™) for 15 min, washed with PBS, incubated with a streptavidin-coupled peroxidase complex (TS-125-HR, LabVision Co™) for 15 min, then washed again with PBS.

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: Ultra V Block (Ultra V Block, TA-125-UB, Fremont, California, USA) was enforced for 5 minutes in order to avoid background staining. .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature.

    Article Title: Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH)
    Article Snippet: After being blocked with Ultra V Block (Thermo Fisher Scientific, Fremont, CA, USA), the slides were incubated with OPN rabbit polyclonal (1:50; cat.no: RB-9097-P0 Labvision, Thermo Fisher Scientific Fremont, CA, USA) and with CD31 rabbit polyclonal (1:100; bs-0468R Woburn, Massachusetts, USA) antibody dilution for 60 minutes at room temperature. .. After being washed with PBS, the sections were treated with streptavidin peroxidase (TS-125-HR, Labvision, Fremont, CA, USA) and then with 3,3’-diaminobenzidine (DAB) chromogen for 30 seconds.

    Article Title: Enhanced Therapeutic Epidermal Growth Factor Receptor (EGFR) Antibody Delivery via Pulsed Ultrasound with Targeting Microbubbles for Glioma Treatment
    Article Snippet: Streptavidin peroxidase (Cat. No. TS-060-HR, Thermo Scientific) reagent was used as the primary antibody to estimate the delivery of injected biotin-modified therapeutic EGFR antibody. .. After blocking, the section was incubated overnight with 100-fold diluted primary antibody and rinsed with PBS.

    Article Title: Enhanced Susceptibility of Ago1/3 Double-Null Mice to Influenza A Virus Infection
    Article Snippet: Endogenous avidin and biotin (catalog number X0590; Dako, Carpinteria, CA) were blocked (10 min each), followed by blocking for 30 min in Superblock (catalog number 37545ZZ; Thermo Scientific Pierce, Rockford, IL). .. Detection of bound secondary antibodies was performed with horseradish peroxidase-labeled streptavidin (catalog number TS-125-HR; Thermo Scientific, Kalamazoo, MI) with 3,3′ diaminobenzidine (DAB; 5 min; catalog number TA-125-HDX; Thermo Shandon) as chromogenic substrate and a light hematoxylin counterstain (catalog number TA-125-MH; Thermo Scientific, Kalamazoo, MI).

    Article Title: ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma
    Article Snippet: For cytoplasmic proteins: following antigen retrieval, slides were first blocked with hydrogren peroxidase block (Thermoscientific, TA-060_H202Q), washed and blocked once more with Ultra V block (Thermoscientific, TA-060-PBQ) to reduce non-specific background. .. Next, slides were washed and incubated in streptavidin solution (Thermoscientific cat. no. TS-060-HR).

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: After washing with PBS for 3 × 5 min, a solution of Ultra V Block (TA-125-UB, Lab Vision Corporation, USA) was applied for 5 min to inhibit background dyeing and incubated at room temperature for 60 min in humidified atmosphere with a 1/200 diluted asprosin primer antibody. .. The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA).

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: In order to prevent dye remaining on the tissues that were washed with PBS for 3 × 5 min, Ultra V Block (TA-125-UB, Lab Vision Corporation, USA) solution was applied for 5 min and they were incubated with 1/200 diluted primary antibody (iris Rabbit Polyclonal H-067-17, Phoenix Pharmaceuticals, Inc., California, USA) at room temperature for 60 min in a humid environment. .. After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS.

    Staining:

    Article Title: Ganoderma lucidum, a promising agent possessing antioxidant and anti-inflammatory effects for treating calvarial defects with graft application in rats 1
    Article Snippet: Paragraph title: Immunohistochemical staining ... The sections were washed 3 times for 5 min in PBS and then were incubated with biotinylated secondary antibody (catalogue #TP-125-BN, Thermo Fisher Scientific, US) for 14 min. After washing with PBS, streptavidin peroxidase (catalogue #TS-125-HR, Thermo Fisher Scientific, US) was applied to the sections for 15 min.

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber. .. Both the intensity and distribution of iNOS, IL-6, L-FABP, NGAL staining were scored in 10 different areas for each samples under light microscope at magnification X400 in the kidney slices.

    Article Title: Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape
    Article Snippet: Collagen deposition was detected using a Masson’s Trichrome stain. .. Treg cells were detected using a rat anti-mouse FoxP3 antibody (14-5773-82, eBioscience, San Diego, CA) with heat induced retrieval, pH 6.0 Target Retrieval Buffer (S699, DAKO, Carpinteria, CA) followed by a horseradish peroxidase labeled streptavidin detection system (TS-125-HR, Thermo Shandon, Pittsburgh, PA).

    Article Title: Immunoexpression of cyclooxygenase-2 in primary gastric carcinomas and lymph node metastases
    Article Snippet: Endogenous peroxide was blocked with a 3% H2 O2 solution in methanol for 10 min, and for unmasking antigens, a retrieval solution (Vector Co™) 1% in hot water (98 °C) was added and incubated for 20 min. Ultra V block (TA-125-UB, LabVision Co™) was added to the sample and incubated for 10 min to inhibit nonspecific background staining. .. A rabbit monoclonal antibody against human COX-2 (Clone SP21, pm 70 kDa, LabVision Co™, 1:200) was applied for 60 min. After washing with phosphate buffered saline (PBS), the sections were incubated with a secondary anti-polyvalent biotinylated goat antibody (TP-125-BN, LabVision Co™) for 15 min, washed with PBS, incubated with a streptavidin-coupled peroxidase complex (TS-125-HR, LabVision Co™) for 15 min, then washed again with PBS.

    Article Title: The effects of morus nigra on the alveolar bone loss in experimentally-induced periodontitis
    Article Snippet: Ultra V Block (Ultra V Block, TA-125-UB, Fremont, California, USA) was enforced for 5 minutes in order to avoid background staining. .. After that, tissues were washed with PBS for 3×5 minutes and incubated with streptavidin peroxidase (Streptavidin Peroxidase, Thermo Fisher Scientific, TS-060-HR, Fremont, California, USA) in a humid environment for 30 minutes at room temperature.

    Western Blot:

    Article Title: The Spermatogonial Stem Cell Niche in the Collared Peccary (Tayassu tajacu) 1
    Article Snippet: Paragraph title: Western Blot Analysis ... The strips were washed three times with TBS-0.05% Tween solution, 5 min each, and incubated in streptavidin solution (TS-125-HR; Thermo Scientific) for 15 min, at room temperature.

    Article Title: Spermatogonial Stem Cell Markers and Niche in Equids
    Article Snippet: Paragraph title: Western blotting analysis ... Before exposing the strips to streptavidin solution (Thermo Scientific, TS-125-HR) for 15 minutes at room temperature, they were rinsed three times with TBS 0.05% Triton-X buffer.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Epidermal growth factor (EGF) promotes bone healing in surgically induced osteonecrosis of the femoral head (ONFH)
    Article Snippet: After being blocked with Ultra V Block (Thermo Fisher Scientific, Fremont, CA, USA), the slides were incubated with OPN rabbit polyclonal (1:50; cat.no: RB-9097-P0 Labvision, Thermo Fisher Scientific Fremont, CA, USA) and with CD31 rabbit polyclonal (1:100; bs-0468R Woburn, Massachusetts, USA) antibody dilution for 60 minutes at room temperature. .. After being washed with PBS, the sections were treated with streptavidin peroxidase (TS-125-HR, Labvision, Fremont, CA, USA) and then with 3,3’-diaminobenzidine (DAB) chromogen for 30 seconds.

    Binding Assay:

    Article Title: Ascorbic acid improves renal microcirculatory oxygenation in a rat model of renal I/R injury
    Article Snippet: Kidney sections were incubated overnight at 4°C with rabbit polyclonal iNOS antibody (iNOS Ab-1, Rabbit PAb, RB-1605-P, NeoMarkers Fremont, CA) and IL-6 (Abcam, 6672), and incubated for 1 h at room temperature with anti-myeloperoxidase (MPO) antibodies (Myeloperoxidase Ab-1, RB-373-A, NeoMarkers Fremont, CA), Lipocalin 2 antibody (NGAL, neutrophil gelatinase-associated lipocalin) (abcam 41105), polyclonal antibody to rat L-FABP, liver-fatty acid binding protein (HycultBiotect HP8010). .. After slides were washed in PBS, the streptavidin peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room temperature in a humid chamber.

    Article Title: Effects of Electrochemotherapy with Cisplatin and Peritumoral IL-12 Gene Electrotransfer on Canine Mast Cell Tumors: a Histopathologic and Immunohistochemical Study
    Article Snippet: .. Antibody binding was detected by the Biotinylated Goat Polyvalent Secondary (TP-125-BN; Thermo Scientific, Cheshire, UK), Streptavidin Peroxidase (TS-125-HR; Thermo Scientific, Cheshire, UK) and DAB chromogen (SK-4105; ImmPact DAB, Vector, Burlingame, CA) as indicated by manufacture’s instructions and slides were counterstained with haematoxylin. .. Serial sections of canine lymph node were used as positive control.

    Microscopy:

    Article Title: Saliva and Blood Asprosin Hormone Concentration Associated with Obesity
    Article Snippet: The tissues were washed with PBS for 3 × 5 min after the application of the secondary antibody and incubated at room temperature for 30 min with Streptavidin Peroxidase (TS-125-HR, Lab Vision Corporation, USA). .. Slides were photographed and examined with a Leica DM500 microscope (Leica DFC295).

    Article Title: Protective effects of benfotiamine on irisin activity in methotrexate-induced liver injury in rats
    Article Snippet: After the application of primary antibody, the tissues were incubated at room temperature for 30 min in a humid environment with a secondary antibody (biotinylated Goat Anti-Polyvalent (anti-mouse/rabbit IgG), TP-125-BN, Lab Vision Corporation, USA) after being washed with PBS for 3 × 5 min. After the application of secondary antibody, the tissues were washed with PBS for 3 × 5 min and after being incubated at room temperature for 30 min in a humid environment with streptavidin peroxidase (TS-125-HR, LabVision Corporation, USA), were placed in PBS. .. Prepared slides were evaluated and photographed on a Leica DM500 microscope (Leica DFC295).

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  • 99
    Thermo Fisher horseradish peroxidase conjugated streptavidin
    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated <t>streptavidin.</t> ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P
    Horseradish Peroxidase Conjugated Streptavidin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/horseradish peroxidase conjugated streptavidin/product/Thermo Fisher
    Average 99 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    horseradish peroxidase conjugated streptavidin - by Bioz Stars, 2020-03
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    99
    Thermo Fisher streptavidin hrp
    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by <t>streptavidin-HRP</t> by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.
    Streptavidin Hrp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin hrp/product/Thermo Fisher
    Average 99 stars, based on 162 article reviews
    Price from $9.99 to $1999.99
    streptavidin hrp - by Bioz Stars, 2020-03
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    Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 stimulates the endocytosis of PrP C . SH-SY5Y cells expressing wild type PrP C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP C . Cells were then lysed and total PrP C immunoprecipitated from the sample using antibody 3F4. ( A ) Samples were subjected to western blot analysis and the biotin-labelled PrP C fraction was detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. ( C ) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP C in the cell lysates from (A). β-actin was used as a loading control. ( D ) SH-SY5Y cells expressing PrP C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( E ) Densitometric analysis of the proportion of total PrP C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. ( F ) SH-SY5Y cells expressing PrP C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Immunoprecipitation, Western Blot, Gradient Centrifugation, Microscopy

    Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 does not affect cell division or surface levels of PrP C . ( A ) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. ( B ) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP Sc ) were detected by immunoblotting using antibody 6D11. ( C ) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Transfection, Incubation, Staining, Fluorescence, Immunoprecipitation

    Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Heparin stimulates the endocytosis of PrP C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts. ( A ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP C . Cells were then lysed and PrP C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP C detected with peroxidase-conjugated streptavidin. ( B ) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. ( C ) SH-SY5Y cells expressing PrP C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( D ) Densitometric analysis of the proportion of total PrP C in the detergent soluble fractions of the plasma membrane. ( E ) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP C using antibody 3F4, with β-actin used as a loading control. ( F ) Quantification of PrP C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot, Gradient Centrifugation, SDS-Gel

    Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: Depletion of glypican-1 inhibits the association of PrP-TM with DRMs. SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. ( A ) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. ( B ) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Expressing, Incubation, Blocking Assay, Homogenization, Gradient Centrifugation, Electrophoresis, Immunoprecipitation, Western Blot

    The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Journal: PLoS Pathogens

    Article Title: Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

    doi: 10.1371/journal.ppat.1000666

    Figure Lengend Snippet: The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC. SH-SY5Y cells expressing PrP-TM were surface biotinylated and then ( A ) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or ( B ) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. ( C ) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P

    Article Snippet: Where indicated, biotin-labelled PrP was detected by subsequent immunoprecipitation of epitope-tagged PrP from the individual fractions using antibody 3F4 (Eurogentec Ltd., Southampton, U.K.) and subsequent immunoblotting using horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Cramlington, U.K.).

    Techniques: Planar Chromatography, Expressing, Incubation, Gradient Centrifugation, Immunoprecipitation, Western Blot

    Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Journal: BMC Cell Biology

    Article Title: A morphologic and semi-quantitative technique to analyze synthesis and release of specific proteins in cells

    doi: 10.1186/s12860-014-0045-1

    Figure Lengend Snippet: Schematic diagram showed non-radioactive metabolic incorporation followed by azide-biotin or azide-Alex555 labeling, and biotin signals of proteins were detected by streptavidin-HRP by western blot. HPG is incorporated into newly synthesized proteins by metabolism and protein synthesis and the triazole conjugation between newly alkyne proteins labeled HPG and azide labeled either biotin or Alex555 via CuSO4 catalysis (A) . (B-a) The detection of biotin signals from extracted total proteins labeled by labeling reaction. Normal culture medium was changed to replace DMEM free of L-methionine supplemented with HPG after pulse 4 hr, and proteins were extracted in each of group at various time points including 0, 4, 24 and 72 hr. (B-b) Biotin signals of total proteins were detected. 1: Normal culture condition group; 2: HPG plus anisomycin group; 3: HPG group. (B-c,d,e) Biotin signals of Bcl-2, MMP-9 and IgG were individually detected in the immunoprecipitate pulled down by primary antibodies via siRNA post-transfection followed by non-radioactive metabolic labeling. (B-f) Radioactive isotope 35 S-methonine incorporated into synthesized IgG purified by immunoprecipitation was detected by autoradiography. 1: 35 S-methonine treated human choriocarcinoma cell line BeWo group and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 2: cycloheximide plus 35 S-methonine treated BeWo group then antibody against human IgG immunoprecipitated human IgG in extracted total proteins; 3: 35 S-methonine treated skin fibroblast and then antibody against human IgG immunoprecipitated human IgG in extracted total proteins.

    Article Snippet: Each diluted standard was incubated with primary antibody coated on the well and streptavidin-HRP and OD value for each sample was detected by ELISA.

    Techniques: Labeling, Western Blot, Synthesized, Conjugation Assay, Transfection, Purification, Immunoprecipitation, Autoradiography

    Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.

    Journal: The Journal of Biological Chemistry

    Article Title: PA1b Inhibitor Binding to Subunits c and e of the Vacuolar ATPase Reveals Its Insecticidal Mechanism *

    doi: 10.1074/jbc.M113.541250

    Figure Lengend Snippet: Electron microscopy of PA1b-bound V-ATPase. A , representative classes of PA1b-streptavidin-HRP-bound V-ATPase in the absence of ATP. B , as A but in the presence of 2 m m Mg·ATP. The PA1b-streptavidin-HRP density is indicated by an arrow in the far left panel 1 of A. Scale bars in both A and B represent 15 nm. C–E , three-dimensional reconstructions of the V-ATPase viewed perpendicular to the long axis of the complex ( upper image ) and from the extracellular end ( lower image ) bound to PA1b ( C ), bound to PA1b after the addition of Mg·ATP ( D ) and a control with no PA1b ( E ). All models were generated using EMAN, and the picture was produced using Chimera rendered at the same sigma level. In C ( lower ), the decameric c ring (Protein Data Bank ID code 2DB4 ( 53 ) r ainbow colors ) and a subunit model ( red ) have been fitted to the PA1b-streptavidin-HRP V-ATPase reconstruction in the absence of ATP using Chimera. If catalytically active, the c ring would rotate counterclockwise with respect to subunit a when observed from this perspective.

    Article Snippet: For the second experiment, 4 μl of V-ATPase (4 μg) was mixed with 3 μl of biotin-PA1b (3 μg) and 3 μl of streptavidin-HRP (15 μg), made up to 60 μl using V-ATPase buffer and incubated for 30 min. Mg·ATP was from a stock solution of 100 mm at pH 7.5 to a final concentration of 5 mm , and the mixture was incubated at room temperature for 5 min to allow for complete turnover.

    Techniques: Electron Microscopy, Generated, Produced

    Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Journal: Veterinary Research

    Article Title: Porcine monocyte subsets differ in the expression of CCR2 and in their responsiveness to CCL2

    doi: 10.1051/vetres/2010048

    Figure Lengend Snippet: Expression of recombinant porcine CCL2. (A) CHO cell line stably expressing the porcine CCL2 fused to GFP. The expression of GFP fusion protein was directly analysed by flow cytometry. Non transfected CHO cells were used as negative control (grey histogram). 5 000 cells were acquired. (B) Western blot of CCL2-GFP produced by transfected CHO cells. Different dilutions of supernatant were resolved by 15% SDS-PAGE under reducing conditions and revealed with biotinylated anti-GFP and streptavidin-HRP. Numbers on the left indicate the position of MW markers. (C) Chemotactic activity of CCL2-GFP on porcine blood monocytes. Chemotaxis was assessed with the Transwell cell migration system and subsequent flow cytometry counting of migrated cells by a 45 s acquisition. (1) FSC versus SSC dot plot of migrated cells in response to supernatants from CHO cells expressing CCL2-GFP or the inverted sequence of pCCL2 fused to GFP (InvCCL2-GFP, negative control). (2) Results expressed as migration index, calculated as the ratio of the number of cells migrating to the chemokine and the number of cells in the negative control. Results from one representative experiment out of three performed are shown. (A color version of this figure is available at www.vetres.org. )

    Article Snippet: The expression of GFP-fused proteins in these clones was confirmed by Western blot using a biotin-conjugated goat anti-GFP polyclonal antibody and streptavidin-HRP.

    Techniques: Expressing, Recombinant, Stable Transfection, Flow Cytometry, Cytometry, Transfection, Negative Control, Western Blot, Produced, SDS Page, Activity Assay, Chemotaxis Assay, Migration, Sequencing