fluorescein antibody  (Abcam)

 
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    Name:
    Anti Fluorescein antibody 6A4
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    Catalog Number:
    ab6213
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    Structured Review

    Abcam fluorescein antibody

    https://www.bioz.com/result/fluorescein antibody/product/Abcam
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fluorescein antibody - by Bioz Stars, 2020-02
    90/100 stars

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    Filtration:

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. For RFP-tagged Agd3 (Agd3-RFP) immunoblotting, mycelia of the wild-type and Agd3-RFP strains were grown at 37°C for 22 to 24 h under shaking conditions, and culture supernatants were recovered by filtration.

    Immunostaining:

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: Phenotypic characterization of fibroblasts Cells cultured from the palatal mucosa and gingival graft were characterized as fibroblasts by their morphology and staining with fibroblast surface protein (FSP; ab 11333, Abcam, Cambridge, UK) by means of immunostaining [ ]. .. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour.

    Incubation:

    Article Title: Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells
    Article Snippet: .. Fluorescein-labeled secondary antibodies (Alexa Fluor488, Alexa Fluor647, 1:1000; Abcam) were applied for 2 h after which the cells were incubated with 0.1% DAPI for 5 min and washed with PBS. .. Microphotographs of LC3 and p-ERK fluorescence were captured on a wide-field fluorescent microscope.

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: .. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour. .. Coverslips were mounted with mounting medium VECTASHIELD Hard-Set Mounting Medium containing 49,6-diamidino-2-Phenylindole (DAPI; H-1500, Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscope laser scanning (TCS model, SPE, Leica Mannheim, Germany).

    Article Title: Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats. Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats
    Article Snippet: .. After washing with phosphate‐buffered saline, the sections were incubated for 1 hour with goat anti‐rabbit IgG as fluorescein secondary antibody (1:50, Alexa Fluor 488; Abcam). .. Finally, the slides were mounted with SlowFade Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA).

    Article Title: Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration
    Article Snippet: .. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-BSP (1:1,000, Abcam), anti-OPN (1:1,000, Abcam), anti-Ocn (1:1,000, Abcam), and anti-β-actin (1:3,000, Abcam) at 4°C overnight and then incubated for 1 hour with fluorescein-conjugated secondary antibodies (Abcam) at RT. .. Protein expression images were visualized using Odyssey V 3.0 image scanning (LI-COR Biosciences, Lincoln, NE, USA).

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. The biomass was crushed under liquid nitrogen, homogenized, and incubated for 1 h at 4°C in the presence of protease inhibitors (Bioshop, Inc.) and Triton X-100 at a final concentration of 2% (Bioshop, Inc.) prior to lyophilization.

    Article Title: Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production
    Article Snippet: .. Subsequently, the sections were incubated for an hour at ambient temperature with fluorescein-conjugated secondary antibodies: Alexa Fluor 488-conjugated goat anti-Syrian hamster IgG, 4 μg/mL (ab180063, Abcam); or Alexa Fluor 594-conjugated donkey anti-rabbit IgG, 4 μg/mL (ab150064, RRID:AB_2734146; Abcam). .. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan).

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: Twofold dilutions of (1S, 3R)-RSL3-fluorescein probe (highest concentration 800 ng) were incubated with 1.6 µg of 6xHis-GPX4U46C protein in each microtube for 30 min at room temperature. .. The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam).

    Article Title: Regulation of DMT1 on autophagy and apoptosis in osteoblast
    Article Snippet: Immunofluorescence Cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 for 5 min. After washing with PBS, secions were incubated in a blocking buffer containing 5% BSA for 30 min at room temperature, followed by incubation with anti-LC3 (1:200) and anti-DMT1(1:200)antibody overnight at 4 ˚C. .. Secondary antibodies labeled with fluorescein (1:500, Abcam, USA) were applied for 120 min. After incubating with 0.1% DAPI for 5 min and another washing step with PBS, coverslips were transferred onto glass slides.

    Article Title: Platelet-derived Growth Factor (PDGF) Regulates Slingshot Phosphatase Activity via Nox1-dependent Auto-dephosphorylation of Serine 834 in Vascular Smooth Muscle Cells
    Article Snippet: After the incubation period, β-mercaptoethanol to a final concentration of 20 m m was added to stop the reaction. .. The lysates were subjected to overnight immunoprecipitation using 5 μg of fluorescein antibody (ab6213, abcam).

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: Cells were washed with 10 mM PBS (pH 7.4) and permeabilized with 0.2% Triton X-100 for 5 min. After washing with 10 mM PBS (pH 7.4), cells were incubated in a blocking buffer containing 5% BSA for 30 min at 20 °C, followed by incubation with anti-MAP2 (1:500 dilution; Proteintech, Chicago, IL, USA), anti-CD133 (1:500 dilution; Abcam), and anti-nestin (1:500 dilution; Abcam) antibodies overnight at 4 °C. .. Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature.

    Article Title: Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue
    Article Snippet: .. After fixation with 4% paraformaldehyde for 15 min, the cells were incubated with 3% bovine serum albumin (BSA) for 30 min at 21°C, then incubated with diluted primary antibody (1:100) anti-fibroblast surface protein (FSP, Abcam) or with a mouse immunoglobulin (IgG1) as a negative control overnight at 40°C, and then finally incubated with fluorescein-conjugated secondary antibody (1:400, Abcam PLC, Cambridge, U.K.) at 37°C for 1 h in the dark. .. Afterwards, the slides were mounted with a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then analyzed (Leica TCS SPE confocal laser scanning microscope, Mannheim, Germany).

    Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
    Article Snippet: .. Upon binding equilibrium, cells were pelleted, washed with PBSA and incubated with fluorescein-conjugated anti-His6 antibody is PBSA. .. Cells were pelleted, washed in PBSA again then analyzed via flow cytometry on an Accuri C6.

    Oxidation Assay:

    Article Title: Platelet-derived Growth Factor (PDGF) Regulates Slingshot Phosphatase Activity via Nox1-dependent Auto-dephosphorylation of Serine 834 in Vascular Smooth Muscle Cells
    Article Snippet: Then, the 5-IAF oxidation assay was performed by adding a 5-IAF solution to the cell lysate to a final concentration of 10 μ m . .. The lysates were subjected to overnight immunoprecipitation using 5 μg of fluorescein antibody (ab6213, abcam).

    Activity Assay:

    Article Title: Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats. Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats
    Article Snippet: After washing with phosphate‐buffered saline, the sections were incubated for 1 hour with goat anti‐rabbit IgG as fluorescein secondary antibody (1:50, Alexa Fluor 488; Abcam). .. To identify osteoclasts, tartrate‐resistant acid phosphatase (TRAP) activity was detected using the TRAP/ALP Kit (Wako Pure Chemical Industries Co., Osaka, Japan).

    Expressing:

    Article Title: Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration
    Article Snippet: After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-BSP (1:1,000, Abcam), anti-OPN (1:1,000, Abcam), anti-Ocn (1:1,000, Abcam), and anti-β-actin (1:3,000, Abcam) at 4°C overnight and then incubated for 1 hour with fluorescein-conjugated secondary antibodies (Abcam) at RT. .. Protein expression images were visualized using Odyssey V 3.0 image scanning (LI-COR Biosciences, Lincoln, NE, USA).

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: Bacterial expression vector pOE30-c-GPX4U46C was a generous gift from Hartmut Kuhn, University Medicine Berlin–Charité, Berlin. .. The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam).

    BIA-KA:

    Article Title: Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration
    Article Snippet: In brief, cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, People’s republic of China), supplemented with 1 nM phenylmethanesulfonyl fluoride (PMSF) (Invitrogen), then the collected protein contents were determined using a BCA protein assay kit (Thermo Fisher Scientific). .. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-BSP (1:1,000, Abcam), anti-OPN (1:1,000, Abcam), anti-Ocn (1:1,000, Abcam), and anti-β-actin (1:3,000, Abcam) at 4°C overnight and then incubated for 1 hour with fluorescein-conjugated secondary antibodies (Abcam) at RT.

    Western Blot:

    Article Title: Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration
    Article Snippet: Paragraph title: Western blot analysis ... After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-BSP (1:1,000, Abcam), anti-OPN (1:1,000, Abcam), anti-Ocn (1:1,000, Abcam), and anti-β-actin (1:3,000, Abcam) at 4°C overnight and then incubated for 1 hour with fluorescein-conjugated secondary antibodies (Abcam) at RT.

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: .. The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam). .. Two million HT-1080 cells were seeded in 10-cm culture dishes.

    Blocking Assay:

    Article Title: Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells
    Article Snippet: After washing with PBS, the cells were permeabilized with 0.2% Triton X-100 for 5 min. After washing with PBS, sections were incubated in a blocking buffer containing 5% BSA for 30 min at room temperature, followed by incubations with anti-LC3 (1:200) and anti-p-ERK (1:500) antibodies overnight at 4 °C. .. Fluorescein-labeled secondary antibodies (Alexa Fluor488, Alexa Fluor647, 1:1000; Abcam) were applied for 2 h after which the cells were incubated with 0.1% DAPI for 5 min and washed with PBS.

    Article Title: Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats. Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats
    Article Snippet: Then, the sections were incubated with Dako protein block solution (Dako, Carpinteria, CA) for 20 minutes to block nonspecific staining. .. After washing with phosphate‐buffered saline, the sections were incubated for 1 hour with goat anti‐rabbit IgG as fluorescein secondary antibody (1:50, Alexa Fluor 488; Abcam).

    Article Title: Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration
    Article Snippet: .. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-BSP (1:1,000, Abcam), anti-OPN (1:1,000, Abcam), anti-Ocn (1:1,000, Abcam), and anti-β-actin (1:3,000, Abcam) at 4°C overnight and then incubated for 1 hour with fluorescein-conjugated secondary antibodies (Abcam) at RT. .. Protein expression images were visualized using Odyssey V 3.0 image scanning (LI-COR Biosciences, Lincoln, NE, USA).

    Article Title: Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production
    Article Snippet: The specimens were blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) for 10 minutes and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-S1P3 antibody, 10 μg/mL (PA5-77744, RRID: AB_2735752; Thermo Fisher Scientific, Waltham, Massachusetts, USA); Syrian hamster anti-podoplanin/gp36 antibody, 8 μg/mL (ab11936, RRID:AB_298718; Abcam, Cambridge, UK); rabbit IgG, 10 μg/mL (GTX35035, RRID:AB_10623175; GeneTex, Irvine, California, USA); or Syrian hamster IgG, 8 μg/mL (ab18426, Abcam). .. Subsequently, the sections were incubated for an hour at ambient temperature with fluorescein-conjugated secondary antibodies: Alexa Fluor 488-conjugated goat anti-Syrian hamster IgG, 4 μg/mL (ab180063, Abcam); or Alexa Fluor 594-conjugated donkey anti-rabbit IgG, 4 μg/mL (ab150064, RRID:AB_2734146; Abcam).

    Article Title: Regulation of DMT1 on autophagy and apoptosis in osteoblast
    Article Snippet: Immunofluorescence Cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After washing with PBS, cells were permeabilized with 0.2% Triton X-100 for 5 min. After washing with PBS, secions were incubated in a blocking buffer containing 5% BSA for 30 min at room temperature, followed by incubation with anti-LC3 (1:200) and anti-DMT1(1:200)antibody overnight at 4 ˚C. .. Secondary antibodies labeled with fluorescein (1:500, Abcam, USA) were applied for 120 min. After incubating with 0.1% DAPI for 5 min and another washing step with PBS, coverslips were transferred onto glass slides.

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: Cells were washed with 10 mM PBS (pH 7.4) and permeabilized with 0.2% Triton X-100 for 5 min. After washing with 10 mM PBS (pH 7.4), cells were incubated in a blocking buffer containing 5% BSA for 30 min at 20 °C, followed by incubation with anti-MAP2 (1:500 dilution; Proteintech, Chicago, IL, USA), anti-CD133 (1:500 dilution; Abcam), and anti-nestin (1:500 dilution; Abcam) antibodies overnight at 4 °C. .. Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature.

    Immunohistochemistry:

    Article Title: Mesenchymal-endothelial-transition contributes to cardiac neovascularization
    Article Snippet: .. Immunohistochemistry and histology, confocal imaging and quantitation, super-resolution microscopy Immunofluorescent staining on frozen sections (7μm) was performed using primary antibodies to VECAD (Catalog#ab33168, Abcam), eNOS (Catalog#ab66127, Abcam), Claudin 5(Catalog#ab53765, Abcam), Occludin (Catalog#ab31721, Abcam), gamma H2AX(Catalog#ab2893, Abcam), p53 (Catalog#ab31333 & ab26, Abcam), Col1 (Catalog#ab6308, Abcam), Podoplanin(Catalog#ab11936, Abcam), alpha-SMA (Catalog#ab5694, Abcam), CD68(Catalog#ab125212, Abcam), CD146 (Catalog#ab75769, Abcam), Cre (Catalog#BIOT-106L & PRB-106P,Covance), Troponin(Catalog#SC-8121, Santa Cruz), NG2 (Catalog# AB5320, EMD Millipore) and associated APC or Fluorescein conjugated secondary antibodies (Abcam, Millipore, Invitrogen) as per manufacturer instructions. .. Labeled sections were imaged using a Leica SP2 AOBS Upright Laser Scanning Confocal Microscope (Leica Microsystems).

    Article Title: Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats. Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats
    Article Snippet: To confirm the presence of inflammatory cells, the pan‐leukocyte marker CD45 was stained by immunohistochemistry. .. After washing with phosphate‐buffered saline, the sections were incubated for 1 hour with goat anti‐rabbit IgG as fluorescein secondary antibody (1:50, Alexa Fluor 488; Abcam).

    Concentration Assay:

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: .. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour. .. Coverslips were mounted with mounting medium VECTASHIELD Hard-Set Mounting Medium containing 49,6-diamidino-2-Phenylindole (DAPI; H-1500, Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscope laser scanning (TCS model, SPE, Leica Mannheim, Germany).

    Article Title: Magnetic-Assisted Cell Alignment within a Magnetic Nanoparticle-Decorated Reduced Graphene Oxide/Collagen 3D Nanocomposite Hydrogel
    Article Snippet: Materials Collagen (type-I, rat tail extract, in 0.02 M acetic acid, initial concentration of 3.35–4 mg·mL−1 ) was procured from BD-Biosciences (Bedford, MA, USA). .. Rabbit anti-tyrosine hydroxylase (TH) and Texas red- and fluorescein-conjugated secondary antibodies were procured from Abcam (Cambridge, UK).

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. The biomass was crushed under liquid nitrogen, homogenized, and incubated for 1 h at 4°C in the presence of protease inhibitors (Bioshop, Inc.) and Triton X-100 at a final concentration of 2% (Bioshop, Inc.) prior to lyophilization.

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: Twofold dilutions of (1S, 3R)-RSL3-fluorescein probe (highest concentration 800 ng) were incubated with 1.6 µg of 6xHis-GPX4U46C protein in each microtube for 30 min at room temperature. .. The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam).

    Article Title: Platelet-derived Growth Factor (PDGF) Regulates Slingshot Phosphatase Activity via Nox1-dependent Auto-dephosphorylation of Serine 834 in Vascular Smooth Muscle Cells
    Article Snippet: After the incubation period, β-mercaptoethanol to a final concentration of 20 m m was added to stop the reaction. .. The lysates were subjected to overnight immunoprecipitation using 5 μg of fluorescein antibody (ab6213, abcam).

    Cell Culture:

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: Phenotypic characterization of fibroblasts Cells cultured from the palatal mucosa and gingival graft were characterized as fibroblasts by their morphology and staining with fibroblast surface protein (FSP; ab 11333, Abcam, Cambridge, UK) by means of immunostaining [ ]. .. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour.

    Imaging:

    Article Title: Mesenchymal-endothelial-transition contributes to cardiac neovascularization
    Article Snippet: .. Immunohistochemistry and histology, confocal imaging and quantitation, super-resolution microscopy Immunofluorescent staining on frozen sections (7μm) was performed using primary antibodies to VECAD (Catalog#ab33168, Abcam), eNOS (Catalog#ab66127, Abcam), Claudin 5(Catalog#ab53765, Abcam), Occludin (Catalog#ab31721, Abcam), gamma H2AX(Catalog#ab2893, Abcam), p53 (Catalog#ab31333 & ab26, Abcam), Col1 (Catalog#ab6308, Abcam), Podoplanin(Catalog#ab11936, Abcam), alpha-SMA (Catalog#ab5694, Abcam), CD68(Catalog#ab125212, Abcam), CD146 (Catalog#ab75769, Abcam), Cre (Catalog#BIOT-106L & PRB-106P,Covance), Troponin(Catalog#SC-8121, Santa Cruz), NG2 (Catalog# AB5320, EMD Millipore) and associated APC or Fluorescein conjugated secondary antibodies (Abcam, Millipore, Invitrogen) as per manufacturer instructions. .. Labeled sections were imaged using a Leica SP2 AOBS Upright Laser Scanning Confocal Microscope (Leica Microsystems).

    Binding Assay:

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: Paragraph title: In vitro binding assay. ... The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam).

    Article Title: Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor
    Article Snippet: .. Upon binding equilibrium, cells were pelleted, washed with PBSA and incubated with fluorescein-conjugated anti-His6 antibody is PBSA. .. Cells were pelleted, washed in PBSA again then analyzed via flow cytometry on an Accuri C6.

    Immunofluorescence:

    Article Title: Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells
    Article Snippet: Paragraph title: Immunofluorescence ... Fluorescein-labeled secondary antibodies (Alexa Fluor488, Alexa Fluor647, 1:1000; Abcam) were applied for 2 h after which the cells were incubated with 0.1% DAPI for 5 min and washed with PBS.

    Article Title: Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production
    Article Snippet: Preparation of fresh-frozen sections and immunofluorescence staining were carried out using Kawamoto’s film method [ ] with some modifications. .. Subsequently, the sections were incubated for an hour at ambient temperature with fluorescein-conjugated secondary antibodies: Alexa Fluor 488-conjugated goat anti-Syrian hamster IgG, 4 μg/mL (ab180063, Abcam); or Alexa Fluor 594-conjugated donkey anti-rabbit IgG, 4 μg/mL (ab150064, RRID:AB_2734146; Abcam).

    Article Title: Regulation of DMT1 on autophagy and apoptosis in osteoblast
    Article Snippet: Paragraph title: Immunofluorescence ... Secondary antibodies labeled with fluorescein (1:500, Abcam, USA) were applied for 120 min. After incubating with 0.1% DAPI for 5 min and another washing step with PBS, coverslips were transferred onto glass slides.

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: After 28 days of culture, positive induction was detected by immunofluorescence staining. .. Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature.

    Article Title: Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue
    Article Snippet: Paragraph title: Immunofluorescence Staining ... After fixation with 4% paraformaldehyde for 15 min, the cells were incubated with 3% bovine serum albumin (BSA) for 30 min at 21°C, then incubated with diluted primary antibody (1:100) anti-fibroblast surface protein (FSP, Abcam) or with a mouse immunoglobulin (IgG1) as a negative control overnight at 40°C, and then finally incubated with fluorescein-conjugated secondary antibody (1:400, Abcam PLC, Cambridge, U.K.) at 37°C for 1 h in the dark.

    Nucleic Acid Electrophoresis:

    Article Title: Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration
    Article Snippet: Equal amounts of cell lysates were loaded in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene fluoride membrane (0.22 μm; EMD Millipore, Billerica, MA, USA). .. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-BSP (1:1,000, Abcam), anti-OPN (1:1,000, Abcam), anti-Ocn (1:1,000, Abcam), and anti-β-actin (1:3,000, Abcam) at 4°C overnight and then incubated for 1 hour with fluorescein-conjugated secondary antibodies (Abcam) at RT.

    Fluorescence:

    Article Title: Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells
    Article Snippet: Fluorescein-labeled secondary antibodies (Alexa Fluor488, Alexa Fluor647, 1:1000; Abcam) were applied for 2 h after which the cells were incubated with 0.1% DAPI for 5 min and washed with PBS. .. Microphotographs of LC3 and p-ERK fluorescence were captured on a wide-field fluorescent microscope.

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: .. To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. The cells were then mounted on microscope slides and imaged by confocal microscopy as previously described ( ).

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: The morphology of the BMSCs and the levels of specific neural markers, including microtubule-associated protein 2 (MAP2), CD133 and nestin in each group were observed using a BX41 upright fluorescence microscope (Olympus). .. Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature.

    Mutagenesis:

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: .. To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. The cells were then mounted on microscope slides and imaged by confocal microscopy as previously described ( ).

    Microscopy:

    Article Title: Mesenchymal-endothelial-transition contributes to cardiac neovascularization
    Article Snippet: .. Immunohistochemistry and histology, confocal imaging and quantitation, super-resolution microscopy Immunofluorescent staining on frozen sections (7μm) was performed using primary antibodies to VECAD (Catalog#ab33168, Abcam), eNOS (Catalog#ab66127, Abcam), Claudin 5(Catalog#ab53765, Abcam), Occludin (Catalog#ab31721, Abcam), gamma H2AX(Catalog#ab2893, Abcam), p53 (Catalog#ab31333 & ab26, Abcam), Col1 (Catalog#ab6308, Abcam), Podoplanin(Catalog#ab11936, Abcam), alpha-SMA (Catalog#ab5694, Abcam), CD68(Catalog#ab125212, Abcam), CD146 (Catalog#ab75769, Abcam), Cre (Catalog#BIOT-106L & PRB-106P,Covance), Troponin(Catalog#SC-8121, Santa Cruz), NG2 (Catalog# AB5320, EMD Millipore) and associated APC or Fluorescein conjugated secondary antibodies (Abcam, Millipore, Invitrogen) as per manufacturer instructions. .. Labeled sections were imaged using a Leica SP2 AOBS Upright Laser Scanning Confocal Microscope (Leica Microsystems).

    Article Title: Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells
    Article Snippet: Fluorescein-labeled secondary antibodies (Alexa Fluor488, Alexa Fluor647, 1:1000; Abcam) were applied for 2 h after which the cells were incubated with 0.1% DAPI for 5 min and washed with PBS. .. Microphotographs of LC3 and p-ERK fluorescence were captured on a wide-field fluorescent microscope.

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour. .. Coverslips were mounted with mounting medium VECTASHIELD Hard-Set Mounting Medium containing 49,6-diamidino-2-Phenylindole (DAPI; H-1500, Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscope laser scanning (TCS model, SPE, Leica Mannheim, Germany).

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: .. To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. The cells were then mounted on microscope slides and imaged by confocal microscopy as previously described ( ).

    Article Title: Regulation of DMT1 on autophagy and apoptosis in osteoblast
    Article Snippet: Secondary antibodies labeled with fluorescein (1:500, Abcam, USA) were applied for 120 min. After incubating with 0.1% DAPI for 5 min and another washing step with PBS, coverslips were transferred onto glass slides. .. Images were captured on a wide-field fluorescent microscopy (Olympus, Japan).

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: The morphology of the BMSCs and the levels of specific neural markers, including microtubule-associated protein 2 (MAP2), CD133 and nestin in each group were observed using a BX41 upright fluorescence microscope (Olympus). .. Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature.

    Purification:

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: The 6xHis-GPX4U46C protein was expressed in E. coli and purified according to a published protocol ( ). .. The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam).

    Article Title: Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue
    Article Snippet: After attachment overnight, cells were stimulated by lipopolysaccharide (LPS) from P . gingivalis or E .coli (10 μg/mL) for 24 h. Purified LPS from P . gingivalis (catalog # tlrl-pglps) and E .coli (catalog # tlrl-eklps) from InvivoGen (San Diego, CA, U.S.A.) were used. .. After fixation with 4% paraformaldehyde for 15 min, the cells were incubated with 3% bovine serum albumin (BSA) for 30 min at 21°C, then incubated with diluted primary antibody (1:100) anti-fibroblast surface protein (FSP, Abcam) or with a mouse immunoglobulin (IgG1) as a negative control overnight at 40°C, and then finally incubated with fluorescein-conjugated secondary antibody (1:400, Abcam PLC, Cambridge, U.K.) at 37°C for 1 h in the dark.

    Labeling:

    Article Title: Mesenchymal-endothelial-transition contributes to cardiac neovascularization
    Article Snippet: Immunohistochemistry and histology, confocal imaging and quantitation, super-resolution microscopy Immunofluorescent staining on frozen sections (7μm) was performed using primary antibodies to VECAD (Catalog#ab33168, Abcam), eNOS (Catalog#ab66127, Abcam), Claudin 5(Catalog#ab53765, Abcam), Occludin (Catalog#ab31721, Abcam), gamma H2AX(Catalog#ab2893, Abcam), p53 (Catalog#ab31333 & ab26, Abcam), Col1 (Catalog#ab6308, Abcam), Podoplanin(Catalog#ab11936, Abcam), alpha-SMA (Catalog#ab5694, Abcam), CD68(Catalog#ab125212, Abcam), CD146 (Catalog#ab75769, Abcam), Cre (Catalog#BIOT-106L & PRB-106P,Covance), Troponin(Catalog#SC-8121, Santa Cruz), NG2 (Catalog# AB5320, EMD Millipore) and associated APC or Fluorescein conjugated secondary antibodies (Abcam, Millipore, Invitrogen) as per manufacturer instructions. .. Labeled sections were imaged using a Leica SP2 AOBS Upright Laser Scanning Confocal Microscope (Leica Microsystems).

    Article Title: Regulation of DMT1 on autophagy and apoptosis in osteoblast
    Article Snippet: .. Secondary antibodies labeled with fluorescein (1:500, Abcam, USA) were applied for 120 min. After incubating with 0.1% DAPI for 5 min and another washing step with PBS, coverslips were transferred onto glass slides. .. Images were captured on a wide-field fluorescent microscopy (Olympus, Japan).

    Article Title: Platelet-derived Growth Factor (PDGF) Regulates Slingshot Phosphatase Activity via Nox1-dependent Auto-dephosphorylation of Serine 834 in Vascular Smooth Muscle Cells
    Article Snippet: Paragraph title: Labeling with 5-Iodoacetamido Fluorescein (5-IAF) ... The lysates were subjected to overnight immunoprecipitation using 5 μg of fluorescein antibody (ab6213, abcam).

    Confocal Microscopy:

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. The cells were then mounted on microscope slides and imaged by confocal microscopy as previously described ( ).

    Lysis:

    Article Title: Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration
    Article Snippet: In brief, cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, People’s republic of China), supplemented with 1 nM phenylmethanesulfonyl fluoride (PMSF) (Invitrogen), then the collected protein contents were determined using a BCA protein assay kit (Thermo Fisher Scientific). .. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies: anti-BSP (1:1,000, Abcam), anti-OPN (1:1,000, Abcam), anti-Ocn (1:1,000, Abcam), and anti-β-actin (1:3,000, Abcam) at 4°C overnight and then incubated for 1 hour with fluorescein-conjugated secondary antibodies (Abcam) at RT.

    SDS Page:

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: .. The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam). .. Two million HT-1080 cells were seeded in 10-cm culture dishes.

    Plasmid Preparation:

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour. .. Coverslips were mounted with mounting medium VECTASHIELD Hard-Set Mounting Medium containing 49,6-diamidino-2-Phenylindole (DAPI; H-1500, Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscope laser scanning (TCS model, SPE, Leica Mannheim, Germany).

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: Bacterial expression vector pOE30-c-GPX4U46C was a generous gift from Hartmut Kuhn, University Medicine Berlin–Charité, Berlin. .. The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam).

    Article Title: Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue
    Article Snippet: After fixation with 4% paraformaldehyde for 15 min, the cells were incubated with 3% bovine serum albumin (BSA) for 30 min at 21°C, then incubated with diluted primary antibody (1:100) anti-fibroblast surface protein (FSP, Abcam) or with a mouse immunoglobulin (IgG1) as a negative control overnight at 40°C, and then finally incubated with fluorescein-conjugated secondary antibody (1:400, Abcam PLC, Cambridge, U.K.) at 37°C for 1 h in the dark. .. Afterwards, the slides were mounted with a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then analyzed (Leica TCS SPE confocal laser scanning microscope, Mannheim, Germany).

    Software:

    Article Title: Mesenchymal-endothelial-transition contributes to cardiac neovascularization
    Article Snippet: Immunohistochemistry and histology, confocal imaging and quantitation, super-resolution microscopy Immunofluorescent staining on frozen sections (7μm) was performed using primary antibodies to VECAD (Catalog#ab33168, Abcam), eNOS (Catalog#ab66127, Abcam), Claudin 5(Catalog#ab53765, Abcam), Occludin (Catalog#ab31721, Abcam), gamma H2AX(Catalog#ab2893, Abcam), p53 (Catalog#ab31333 & ab26, Abcam), Col1 (Catalog#ab6308, Abcam), Podoplanin(Catalog#ab11936, Abcam), alpha-SMA (Catalog#ab5694, Abcam), CD68(Catalog#ab125212, Abcam), CD146 (Catalog#ab75769, Abcam), Cre (Catalog#BIOT-106L & PRB-106P,Covance), Troponin(Catalog#SC-8121, Santa Cruz), NG2 (Catalog# AB5320, EMD Millipore) and associated APC or Fluorescein conjugated secondary antibodies (Abcam, Millipore, Invitrogen) as per manufacturer instructions. .. Colocalization analysis of confocal images was performed using Image J software (NIH).

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature. .. The mean fluorescence intensity of MAP2, CD133 and nestin was analysed using ImageJ software (NIH).

    Negative Control:

    Article Title: Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue
    Article Snippet: .. After fixation with 4% paraformaldehyde for 15 min, the cells were incubated with 3% bovine serum albumin (BSA) for 30 min at 21°C, then incubated with diluted primary antibody (1:100) anti-fibroblast surface protein (FSP, Abcam) or with a mouse immunoglobulin (IgG1) as a negative control overnight at 40°C, and then finally incubated with fluorescein-conjugated secondary antibody (1:400, Abcam PLC, Cambridge, U.K.) at 37°C for 1 h in the dark. .. Afterwards, the slides were mounted with a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then analyzed (Leica TCS SPE confocal laser scanning microscope, Mannheim, Germany).

    In Vitro:

    Article Title: Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis
    Article Snippet: Paragraph title: In vitro binding assay. ... The resulting samples were resolved on SDS/PAGE followed by Western blotting with either antifluorescein antibody or anti-GPX4 antibody (ab41787; Abcam).

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: Paragraph title: Differentiation of BMSCs into neural-like stem-like cells in vitro ... Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature.

    Laser-Scanning Microscopy:

    Article Title: Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue
    Article Snippet: After fixation with 4% paraformaldehyde for 15 min, the cells were incubated with 3% bovine serum albumin (BSA) for 30 min at 21°C, then incubated with diluted primary antibody (1:100) anti-fibroblast surface protein (FSP, Abcam) or with a mouse immunoglobulin (IgG1) as a negative control overnight at 40°C, and then finally incubated with fluorescein-conjugated secondary antibody (1:400, Abcam PLC, Cambridge, U.K.) at 37°C for 1 h in the dark. .. Afterwards, the slides were mounted with a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then analyzed (Leica TCS SPE confocal laser scanning microscope, Mannheim, Germany).

    Quantitation Assay:

    Article Title: Mesenchymal-endothelial-transition contributes to cardiac neovascularization
    Article Snippet: .. Immunohistochemistry and histology, confocal imaging and quantitation, super-resolution microscopy Immunofluorescent staining on frozen sections (7μm) was performed using primary antibodies to VECAD (Catalog#ab33168, Abcam), eNOS (Catalog#ab66127, Abcam), Claudin 5(Catalog#ab53765, Abcam), Occludin (Catalog#ab31721, Abcam), gamma H2AX(Catalog#ab2893, Abcam), p53 (Catalog#ab31333 & ab26, Abcam), Col1 (Catalog#ab6308, Abcam), Podoplanin(Catalog#ab11936, Abcam), alpha-SMA (Catalog#ab5694, Abcam), CD68(Catalog#ab125212, Abcam), CD146 (Catalog#ab75769, Abcam), Cre (Catalog#BIOT-106L & PRB-106P,Covance), Troponin(Catalog#SC-8121, Santa Cruz), NG2 (Catalog# AB5320, EMD Millipore) and associated APC or Fluorescein conjugated secondary antibodies (Abcam, Millipore, Invitrogen) as per manufacturer instructions. .. Labeled sections were imaged using a Leica SP2 AOBS Upright Laser Scanning Confocal Microscope (Leica Microsystems).

    Immunoprecipitation:

    Article Title: Platelet-derived Growth Factor (PDGF) Regulates Slingshot Phosphatase Activity via Nox1-dependent Auto-dephosphorylation of Serine 834 in Vascular Smooth Muscle Cells
    Article Snippet: .. The lysates were subjected to overnight immunoprecipitation using 5 μg of fluorescein antibody (ab6213, abcam). ..

    Marker:

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: .. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour. .. Coverslips were mounted with mounting medium VECTASHIELD Hard-Set Mounting Medium containing 49,6-diamidino-2-Phenylindole (DAPI; H-1500, Vector Laboratories, Burlingame, CA, USA) and analyzed by confocal microscope laser scanning (TCS model, SPE, Leica Mannheim, Germany).

    Article Title: Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats. Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats
    Article Snippet: To confirm the presence of inflammatory cells, the pan‐leukocyte marker CD45 was stained by immunohistochemistry. .. After washing with phosphate‐buffered saline, the sections were incubated for 1 hour with goat anti‐rabbit IgG as fluorescein secondary antibody (1:50, Alexa Fluor 488; Abcam).

    Staining:

    Article Title: Mesenchymal-endothelial-transition contributes to cardiac neovascularization
    Article Snippet: .. Immunohistochemistry and histology, confocal imaging and quantitation, super-resolution microscopy Immunofluorescent staining on frozen sections (7μm) was performed using primary antibodies to VECAD (Catalog#ab33168, Abcam), eNOS (Catalog#ab66127, Abcam), Claudin 5(Catalog#ab53765, Abcam), Occludin (Catalog#ab31721, Abcam), gamma H2AX(Catalog#ab2893, Abcam), p53 (Catalog#ab31333 & ab26, Abcam), Col1 (Catalog#ab6308, Abcam), Podoplanin(Catalog#ab11936, Abcam), alpha-SMA (Catalog#ab5694, Abcam), CD68(Catalog#ab125212, Abcam), CD146 (Catalog#ab75769, Abcam), Cre (Catalog#BIOT-106L & PRB-106P,Covance), Troponin(Catalog#SC-8121, Santa Cruz), NG2 (Catalog# AB5320, EMD Millipore) and associated APC or Fluorescein conjugated secondary antibodies (Abcam, Millipore, Invitrogen) as per manufacturer instructions. .. Labeled sections were imaged using a Leica SP2 AOBS Upright Laser Scanning Confocal Microscope (Leica Microsystems).

    Article Title: Inhibition of autophagy increased AGE/ROS-mediated apoptosis in mesangial cells
    Article Snippet: Fluorescein-labeled secondary antibodies (Alexa Fluor488, Alexa Fluor647, 1:1000; Abcam) were applied for 2 h after which the cells were incubated with 0.1% DAPI for 5 min and washed with PBS. .. The detection of punctate LC3 staining from the diffuse staining indicated the formation of autophagosomes.

    Article Title: Palatal mucosa derived fibroblasts present an adaptive behavior regarding cytokine secretion when grafted onto the gingival margin
    Article Snippet: Phenotypic characterization of fibroblasts Cells cultured from the palatal mucosa and gingival graft were characterized as fibroblasts by their morphology and staining with fibroblast surface protein (FSP; ab 11333, Abcam, Cambridge, UK) by means of immunostaining [ ]. .. Cells were fixed with paraformaldehyde 4% (15 min) and were washed 3 times with 1x PBS and incubated with PBS BSA 3% (30 min at room temperature) followed by primary monoclonal antibody incubation to fibroblast surface marker diluted at 1:100 (2 μg/mL final concentration), and finally with fluorescein-conjugated secondary antibody (1:400) (Abcam) at 37°C in the dark for 1 hour.

    Article Title: Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats. Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats
    Article Snippet: Then, the sections were incubated with Dako protein block solution (Dako, Carpinteria, CA) for 20 minutes to block nonspecific staining. .. After washing with phosphate‐buffered saline, the sections were incubated for 1 hour with goat anti‐rabbit IgG as fluorescein secondary antibody (1:50, Alexa Fluor 488; Abcam).

    Article Title: Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation
    Article Snippet: .. To determine the localization of Agd3 by fluorescence microscopy, the Af293 agd3-rfp mutant was grown on glass coverslips in RPMI 1640 for 9 to 12 h at 37°C and 5% CO2 , fixed with 4% paraformaldehyde, stained with anti-red fluorescent protein (anti-RFP) IgG antibody (Abcam, Inc.), and detected with fluorescein-tagged anti-IgG antibody (Abcam, Inc.). .. The cells were then mounted on microscope slides and imaged by confocal microscopy as previously described ( ).

    Article Title: Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production
    Article Snippet: Preparation of fresh-frozen sections and immunofluorescence staining were carried out using Kawamoto’s film method [ ] with some modifications. .. Subsequently, the sections were incubated for an hour at ambient temperature with fluorescein-conjugated secondary antibodies: Alexa Fluor 488-conjugated goat anti-Syrian hamster IgG, 4 μg/mL (ab180063, Abcam); or Alexa Fluor 594-conjugated donkey anti-rabbit IgG, 4 μg/mL (ab150064, RRID:AB_2734146; Abcam).

    Article Title: Lentiviral-mediated ephrin B2 gene modification of rat bone marrow mesenchymal stem cells
    Article Snippet: After 28 days of culture, positive induction was detected by immunofluorescence staining. .. Secondary antibodies labelled with fluorescein (1:500 dilution, Abcam) were applied for 120 min at room temperature.

    Article Title: Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue
    Article Snippet: Paragraph title: Immunofluorescence Staining ... After fixation with 4% paraformaldehyde for 15 min, the cells were incubated with 3% bovine serum albumin (BSA) for 30 min at 21°C, then incubated with diluted primary antibody (1:100) anti-fibroblast surface protein (FSP, Abcam) or with a mouse immunoglobulin (IgG1) as a negative control overnight at 40°C, and then finally incubated with fluorescein-conjugated secondary antibody (1:400, Abcam PLC, Cambridge, U.K.) at 37°C for 1 h in the dark.

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  • gfp  (Abcam)
    99
    Abcam gfp
    BAY 11-7082 suppresses the LPS- or IL-1-stimulated formation of <t>K63-pUb</t> chains and the DNA damage response ( A and B ) The experiment was carried out as in Figure 2 , except that the K63-pUb chains formed in response to LPS ( A ) or IL-1β ( B ) were captured on Halo-NEMO from 6 mg (RAW cells) or 3 mg (IL-1R cells) of cell extract protein as described in the Experimental section. The K63-pUb chains were identified by immunoblotting with a specific antibody. Further aliquots of the cell extract were immunoblotted for IKKβ phosphorylation, p105 phosphorylation and GAPDH as in Figure 2 . ( C ) Indirect immunofluorescence images of U2OS cells transiently expressing <t>GFP–RAP80[1–200].</t> Cells were incubated for 1 h with or without BAY 11-7082 (15 μM) and either exposed to ionizing radiation (IR) or not exposed. GFP–RAP80 or γH2AX were visualized using anti-GFP and anti-γH2AX antibodies respectively, and nuclei were stained with DAPI.
    Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 566 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp/product/Abcam
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    77
    Abcam phosphorylated ham 5 gfp
    <t>HAM-5-GFP</t> shows oscillatory localization to fusion points and puncta in hyphae showing chemotropism. (A) Time course of HAM-5-GFP localization to interacting hyphae prior to cell fusion. HAM-5-GFP localized to the hyphal tip of a homing hyphae (white arrow T = 0; T = 8), followed by a disappearance and localization of HAM-5-GFP at the cell surface in the receptive hypha (white arrow; T = 4). Red arrow shows localization to septa near fusion points. At T = 30, HAM-5 is observed at the site of contact (white arrow). Bright field image is shown in upper left panel; remaining panels show GFP fluorescence. Scale bar = 50 µM. (B) Graphical representation of relative fluorescence intensity (R.F.I.) of HAM-5-GFP localization to the receptive hypha and the homing hypha over the time course (panel A). × axis shows time (min). (C) Graphical representation of HAM-5-GFP fluorescence of interacting fusion hyphae shown in Figure S6B over an extended time course. Note that following the fusion event (T = 22 min), HAM-5-GFP puncta co-oscillate in both hyphae for an additional 30 minutes, see (D). y axis shows maximal fluorescence intensity (M.F.I.) while the × axis shows time (min). The time points at which the individual pictures taken at T = 4, T = 8, T = 46 and T = 59 minutes are pointed out in the graph (black arrows) ( Figure S6 and Movie S5 ). The time of fusion at T = 22 min is marked with a black line. (D) Example of HAM-5-GFP appearing in puncta in both hyphae after fusion at T = 46.
    Phosphorylated Ham 5 Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd14  (Abcam)
    99
    Abcam cd14
    Physical interaction of SP2 with the <t>Cd14</t> promoter derived from C3Bir. A SP2 electrophoretic mobility shift was performed running a biotin-labelled Sp2 consensus oligonucleotide alone (lane 1) or incubated with a nuclear extracts isolated from RAW264.7 cells (lane 2). In the latter two complex formations (I and II) from the Sp2 consensus oligo with nuclear extract components were detected. Competition of increasing amounts of unlabelled SP2 oligo for binding to the nuclear extract proteins led to a stepwise disappearance of biotin-Sp2-oligo protein complexes (lane 3–5). After pre-incubation of the nuclear extracts with an SP2 specific antibody new complexes (III-V) were established in this supershift assay demonstrating physical interaction of the SP2 protein with its consensus oligo (lane 6). Specific SP2 DNA complex formation was also demonstrated by the failure of SP1 and SP3 antibodies to bind to the protein DNA complexes (lane 7 and 8). B BMMs after four days of culture. The scale bar represents 10μm at a 10x magnification. C The MMULV-mediated overexpression of mouse SP2 in primary BMMs derived from C3Bir mice correlated with a higher Cd14 transcription rate compared to untreated and control virus infected macrophages. ***: p
    Cd14, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam fluorescein isothiocyanate conjugated anti tlr9 ab
    Expression of <t>TLR9</t> on the surface of human neutrophils stimulated with Hp-DNA and H. pylori . Neutrophils (2.5×10 5 ) were incubated in the presence of Hp-DNA (1 µg/mL) or H. pylori (2.5×10 7 bacteria) or unstimulated (control) for 3 h and the TLR9 expression were analyzed by confocal microscopy ( A ) and by flow cytometry ( B–D ). For confocal microscopy, neutrophils were stained with fluorescein <t>isothiocyanate-conjugated</t> anti-TLR9 Ab (green) and nuclei stained with TOTO-3 iodide (red); and for flow cytometry they were staining with FITC-conjugated anti-TLR9 Ab (purple, red and green histograms); and isotype control (blue histogram) and unstained neutrophils were included (black line), the MFI data is shown in B . This figure represents one of three ( A ) and six ( B ) independent experiments performed. The MFI and positive cells in % data are represented as mean + SD of six independent experiments ( C and D respectively).
    Fluorescein Isothiocyanate Conjugated Anti Tlr9 Ab, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    BAY 11-7082 suppresses the LPS- or IL-1-stimulated formation of K63-pUb chains and the DNA damage response ( A and B ) The experiment was carried out as in Figure 2 , except that the K63-pUb chains formed in response to LPS ( A ) or IL-1β ( B ) were captured on Halo-NEMO from 6 mg (RAW cells) or 3 mg (IL-1R cells) of cell extract protein as described in the Experimental section. The K63-pUb chains were identified by immunoblotting with a specific antibody. Further aliquots of the cell extract were immunoblotted for IKKβ phosphorylation, p105 phosphorylation and GAPDH as in Figure 2 . ( C ) Indirect immunofluorescence images of U2OS cells transiently expressing GFP–RAP80[1–200]. Cells were incubated for 1 h with or without BAY 11-7082 (15 μM) and either exposed to ionizing radiation (IR) or not exposed. GFP–RAP80 or γH2AX were visualized using anti-GFP and anti-γH2AX antibodies respectively, and nuclei were stained with DAPI.

    Journal: Biochemical Journal

    Article Title: The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system

    doi: 10.1042/BJ20121651

    Figure Lengend Snippet: BAY 11-7082 suppresses the LPS- or IL-1-stimulated formation of K63-pUb chains and the DNA damage response ( A and B ) The experiment was carried out as in Figure 2 , except that the K63-pUb chains formed in response to LPS ( A ) or IL-1β ( B ) were captured on Halo-NEMO from 6 mg (RAW cells) or 3 mg (IL-1R cells) of cell extract protein as described in the Experimental section. The K63-pUb chains were identified by immunoblotting with a specific antibody. Further aliquots of the cell extract were immunoblotted for IKKβ phosphorylation, p105 phosphorylation and GAPDH as in Figure 2 . ( C ) Indirect immunofluorescence images of U2OS cells transiently expressing GFP–RAP80[1–200]. Cells were incubated for 1 h with or without BAY 11-7082 (15 μM) and either exposed to ionizing radiation (IR) or not exposed. GFP–RAP80 or γH2AX were visualized using anti-GFP and anti-γH2AX antibodies respectively, and nuclei were stained with DAPI.

    Article Snippet: Antibodies that recognize GFP (green fluorescent protein) (Abcam), K63-pUb chains (eBioscience), K48-pUb chains, IRAK4 and histone γH2AX (Merck-Millipore) were purchased from the sources indicated.

    Techniques: Immunofluorescence, Expressing, Incubation, Staining

    HAM-5-GFP shows oscillatory localization to fusion points and puncta in hyphae showing chemotropism. (A) Time course of HAM-5-GFP localization to interacting hyphae prior to cell fusion. HAM-5-GFP localized to the hyphal tip of a homing hyphae (white arrow T = 0; T = 8), followed by a disappearance and localization of HAM-5-GFP at the cell surface in the receptive hypha (white arrow; T = 4). Red arrow shows localization to septa near fusion points. At T = 30, HAM-5 is observed at the site of contact (white arrow). Bright field image is shown in upper left panel; remaining panels show GFP fluorescence. Scale bar = 50 µM. (B) Graphical representation of relative fluorescence intensity (R.F.I.) of HAM-5-GFP localization to the receptive hypha and the homing hypha over the time course (panel A). × axis shows time (min). (C) Graphical representation of HAM-5-GFP fluorescence of interacting fusion hyphae shown in Figure S6B over an extended time course. Note that following the fusion event (T = 22 min), HAM-5-GFP puncta co-oscillate in both hyphae for an additional 30 minutes, see (D). y axis shows maximal fluorescence intensity (M.F.I.) while the × axis shows time (min). The time points at which the individual pictures taken at T = 4, T = 8, T = 46 and T = 59 minutes are pointed out in the graph (black arrows) ( Figure S6 and Movie S5 ). The time of fusion at T = 22 min is marked with a black line. (D) Example of HAM-5-GFP appearing in puncta in both hyphae after fusion at T = 46.

    Journal: PLoS Genetics

    Article Title: HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    doi: 10.1371/journal.pgen.1004783

    Figure Lengend Snippet: HAM-5-GFP shows oscillatory localization to fusion points and puncta in hyphae showing chemotropism. (A) Time course of HAM-5-GFP localization to interacting hyphae prior to cell fusion. HAM-5-GFP localized to the hyphal tip of a homing hyphae (white arrow T = 0; T = 8), followed by a disappearance and localization of HAM-5-GFP at the cell surface in the receptive hypha (white arrow; T = 4). Red arrow shows localization to septa near fusion points. At T = 30, HAM-5 is observed at the site of contact (white arrow). Bright field image is shown in upper left panel; remaining panels show GFP fluorescence. Scale bar = 50 µM. (B) Graphical representation of relative fluorescence intensity (R.F.I.) of HAM-5-GFP localization to the receptive hypha and the homing hypha over the time course (panel A). × axis shows time (min). (C) Graphical representation of HAM-5-GFP fluorescence of interacting fusion hyphae shown in Figure S6B over an extended time course. Note that following the fusion event (T = 22 min), HAM-5-GFP puncta co-oscillate in both hyphae for an additional 30 minutes, see (D). y axis shows maximal fluorescence intensity (M.F.I.) while the × axis shows time (min). The time points at which the individual pictures taken at T = 4, T = 8, T = 46 and T = 59 minutes are pointed out in the graph (black arrows) ( Figure S6 and Movie S5 ). The time of fusion at T = 22 min is marked with a black line. (D) Example of HAM-5-GFP appearing in puncta in both hyphae after fusion at T = 46.

    Article Snippet: Detection of phosphorylated HAM-5-GFP was performed using anti-phosphothreonine-proline/phosphoserine-proline antibodies (Abcam).

    Techniques: Fluorescence

    HAM-5-GFP and MAK-2-mCherry localize to puncta in Δ ham-7 and Δ ham-11 fusion-deficient germlings. (A) Western blot of protein samples from 5 hr-old WT, Δ ham-5 , Δ ham-7 and Δ ham-11 germlings probed with anti-p42/44 antibodies, which recognize phosphorylated MAK-1 and MAK-2 [8] . As previously shown [43] , MAK-1 phosphorylation is reduced in the Δ ham-7 mutant. (B) HAM-5-GFP and MAK-2-mCherry localization in WT germlings undergoing chemotropic interactions. Arrows show localization to the CAT tip and to cytoplasmic puncta. (C) HAM-5-GFP and MAK-2-mCherry co-localization in Δ ham-7 ( ham-5-gfp; mak-2-mCherry ) germlings. Note lack of chemotropic interactions. Arrows show puncta of HAM-5-GFP and MAK-2-mCherry in Δ ham-7 ( ham-5-gfp; mak-2-mCherry ) germlings, which are often co-localized at the germ tube tip. (D) HAM-5-GFP and MAK-2-mCherry co-localization in Δ ham-11 ( ham-5-gfp; mak-2-mCherry ) germlings. Note lack of chemotropic interactions. HAM-5-GFP and MAK-2-mCherry co-localized to both cytoplasmic and tip-localized puncta (arrows). The upper left panels are bright field images, the upper right panels show GFP fluorescence, the lower left panels show mCherry fluorescence. Lower right panels show merged images of GFP and mCherry images. Scale bars = 10 µM.

    Journal: PLoS Genetics

    Article Title: HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    doi: 10.1371/journal.pgen.1004783

    Figure Lengend Snippet: HAM-5-GFP and MAK-2-mCherry localize to puncta in Δ ham-7 and Δ ham-11 fusion-deficient germlings. (A) Western blot of protein samples from 5 hr-old WT, Δ ham-5 , Δ ham-7 and Δ ham-11 germlings probed with anti-p42/44 antibodies, which recognize phosphorylated MAK-1 and MAK-2 [8] . As previously shown [43] , MAK-1 phosphorylation is reduced in the Δ ham-7 mutant. (B) HAM-5-GFP and MAK-2-mCherry localization in WT germlings undergoing chemotropic interactions. Arrows show localization to the CAT tip and to cytoplasmic puncta. (C) HAM-5-GFP and MAK-2-mCherry co-localization in Δ ham-7 ( ham-5-gfp; mak-2-mCherry ) germlings. Note lack of chemotropic interactions. Arrows show puncta of HAM-5-GFP and MAK-2-mCherry in Δ ham-7 ( ham-5-gfp; mak-2-mCherry ) germlings, which are often co-localized at the germ tube tip. (D) HAM-5-GFP and MAK-2-mCherry co-localization in Δ ham-11 ( ham-5-gfp; mak-2-mCherry ) germlings. Note lack of chemotropic interactions. HAM-5-GFP and MAK-2-mCherry co-localized to both cytoplasmic and tip-localized puncta (arrows). The upper left panels are bright field images, the upper right panels show GFP fluorescence, the lower left panels show mCherry fluorescence. Lower right panels show merged images of GFP and mCherry images. Scale bars = 10 µM.

    Article Snippet: Detection of phosphorylated HAM-5-GFP was performed using anti-phosphothreonine-proline/phosphoserine-proline antibodies (Abcam).

    Techniques: Western Blot, Mutagenesis, Fluorescence

    HAM-5 is required to localize MAK-2 and MEK-2 to puncta. (A) MAK-2-GFP in isolated WT germlings localizes to the nucleus, cytoplasm and to puncta (white arrows). (B) In the Δ ham-5 mutant, MAK-2-GFP localization is cytoplasmic and nuclear; no puncta are observed. Scale bar = 10 µM. (C) In WT hyphae, MEK-2-mCherry localizes to the septum (green arrow) and also to cytoplasmic puncta (white arrow). Scale bar = 10 µM. (D) In Δ ham-5 hyphae, septum localization of MEK-2-mCherry is observed (green arrow), but puncta are not. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between MEK-2-mCherry and phosphorylated MAK-2 in WT ( mek-2-mCherry ) germlings, but a significant reduction in interaction between MAK-2 and MEK-2-mCherry in a Δ ham-5 ( mek-2-mCherry ) strain. Top panel is a Western blot of protein samples probed with anti-p42/44 antibodies (MAK-2, 40.8 kD, MAK-1, 46.7 kD; Figure S4E ). Middle panel is anti-mCherry immunoprecipitated proteins probed with anti-mCherry antibodies (MEK-2-mCherry, 82.9 kD; Figure S4C , E). Bottom panel is anti-mCherry (MEK-2-mCherry) immunoprecipitated proteins probed via Western blot with anti-p42/44 antibodies that recognize phosphorylated MAK-2.

    Journal: PLoS Genetics

    Article Title: HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    doi: 10.1371/journal.pgen.1004783

    Figure Lengend Snippet: HAM-5 is required to localize MAK-2 and MEK-2 to puncta. (A) MAK-2-GFP in isolated WT germlings localizes to the nucleus, cytoplasm and to puncta (white arrows). (B) In the Δ ham-5 mutant, MAK-2-GFP localization is cytoplasmic and nuclear; no puncta are observed. Scale bar = 10 µM. (C) In WT hyphae, MEK-2-mCherry localizes to the septum (green arrow) and also to cytoplasmic puncta (white arrow). Scale bar = 10 µM. (D) In Δ ham-5 hyphae, septum localization of MEK-2-mCherry is observed (green arrow), but puncta are not. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between MEK-2-mCherry and phosphorylated MAK-2 in WT ( mek-2-mCherry ) germlings, but a significant reduction in interaction between MAK-2 and MEK-2-mCherry in a Δ ham-5 ( mek-2-mCherry ) strain. Top panel is a Western blot of protein samples probed with anti-p42/44 antibodies (MAK-2, 40.8 kD, MAK-1, 46.7 kD; Figure S4E ). Middle panel is anti-mCherry immunoprecipitated proteins probed with anti-mCherry antibodies (MEK-2-mCherry, 82.9 kD; Figure S4C , E). Bottom panel is anti-mCherry (MEK-2-mCherry) immunoprecipitated proteins probed via Western blot with anti-p42/44 antibodies that recognize phosphorylated MAK-2.

    Article Snippet: Detection of phosphorylated HAM-5-GFP was performed using anti-phosphothreonine-proline/phosphoserine-proline antibodies (Abcam).

    Techniques: Isolation, Mutagenesis, Immunoprecipitation, Western Blot

    HAM-5-GFP shows localization with components of the MAK-2 pathway. (A) Co-localization of HAM-5-GFP and MAK-2-mCherry during germling communication (arrows). (B) Co-localization of HAM-5-GFP and MEK-2-mCherry during germling communication (arrows). (C) Co-localization of HAM-5-GFP and NRC-1-mCherry during germling communication. NRC-1-mCherry strains show low fluorescence [18] . (D) HAM-5-GFP and SO-mCherry do not co-localize during chemotropic interactions, but instead show opposite localization to CAT tips in communicating germlings (arrows). The images on the left are bright field images, fluorescent images on the right. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between HAM-5-GFP (210 kD; Figure S4D ) and MEK-2-mCherry (82.9 kD; Figure S4C ) and NRC-1-mCherry (128 kD; Figure S4C ). Input panels show Western blots of immunoprecipitated protein samples from 5 hr-old germlings probed with either anti-GFP (free GFP = 27 kD; Figure S4D ) or anti-mCherry antibodies. The output panel is a Western blot of proteins immunoprecipitated by anti-GFP antibodies (and thus HAM-5-GFP) and probed with anti-mCherry antibodies (detecting MEK-2-mCherry or NRC-1-mCherry).

    Journal: PLoS Genetics

    Article Title: HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    doi: 10.1371/journal.pgen.1004783

    Figure Lengend Snippet: HAM-5-GFP shows localization with components of the MAK-2 pathway. (A) Co-localization of HAM-5-GFP and MAK-2-mCherry during germling communication (arrows). (B) Co-localization of HAM-5-GFP and MEK-2-mCherry during germling communication (arrows). (C) Co-localization of HAM-5-GFP and NRC-1-mCherry during germling communication. NRC-1-mCherry strains show low fluorescence [18] . (D) HAM-5-GFP and SO-mCherry do not co-localize during chemotropic interactions, but instead show opposite localization to CAT tips in communicating germlings (arrows). The images on the left are bright field images, fluorescent images on the right. Scale bar = 10 µM. (E) Co-immunoprecipitation experiments showing an interaction between HAM-5-GFP (210 kD; Figure S4D ) and MEK-2-mCherry (82.9 kD; Figure S4C ) and NRC-1-mCherry (128 kD; Figure S4C ). Input panels show Western blots of immunoprecipitated protein samples from 5 hr-old germlings probed with either anti-GFP (free GFP = 27 kD; Figure S4D ) or anti-mCherry antibodies. The output panel is a Western blot of proteins immunoprecipitated by anti-GFP antibodies (and thus HAM-5-GFP) and probed with anti-mCherry antibodies (detecting MEK-2-mCherry or NRC-1-mCherry).

    Article Snippet: Detection of phosphorylated HAM-5-GFP was performed using anti-phosphothreonine-proline/phosphoserine-proline antibodies (Abcam).

    Techniques: Fluorescence, Immunoprecipitation, Western Blot

    HAM-5-GFP localization in WT and Δ mak-2 germlings. (A) Schematic overview of HAM-5 protein structure. The predicted WD40 domains are shown in grey and the putative coiled coil domains are shown as red bars. The two disordered regions with low complexity are depicted by shaded white boxes. The MAPK phosphorylation site (aa 506) is marked by a blue star, the other two sites showing decreased abundance in treated cells (aa 1288 and 1604) are marked by green stars, and other 13 identified phosphorylation sites (S14, S414, S792, S818, S833, T838, T969, S1085, S1199, T1201, S1202, T1353, S1608) [40] are marked by black stars. The putative MAPK docking site is marked by a yellow line. (B) Localization of HAM-5-GFP to puncta localized to CAT tips during chemotropic interactions between genetically identical cells. HAM-5-GFP showed dynamic localization to CAT tips of germlings with an oscillation of every four min (arrow). HAM-5-GFP also localized to puncta within germlings and near nuclear compartments devoid of HAM-5-GFP (asterisks). The image left is a bright field image. Scale bar = 10 µM. (C) HAM-5-GFP localized to the sites of contact during germling fusion (arrow). (D) Western blots of WT, WT ( ham-5-gfp ) and Δ mak-2 ( ham-5-gfp ) germlings with immunoprecipitated HAM-5-GFP probed with anti-GFP antibodies (right panel shows longer run showing higher mobility of HAM-5-GFP in wild type germlings) specifically detecting HAM-5-GFP (210 kD; Figure S4D ). Lower panel shows a Western blot with identical samples probed with anti-phospho antibodies that specifically detect phosphorylated serine or threonine residues followed by a proline. (E) Localization of HAM-5-GFP to puncta in Δ mak-2 germlings. Some puncta showed localization to germling tips, but which did not oscillate during growth (white arrows). Scale bar = 10 µM.

    Journal: PLoS Genetics

    Article Title: HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    doi: 10.1371/journal.pgen.1004783

    Figure Lengend Snippet: HAM-5-GFP localization in WT and Δ mak-2 germlings. (A) Schematic overview of HAM-5 protein structure. The predicted WD40 domains are shown in grey and the putative coiled coil domains are shown as red bars. The two disordered regions with low complexity are depicted by shaded white boxes. The MAPK phosphorylation site (aa 506) is marked by a blue star, the other two sites showing decreased abundance in treated cells (aa 1288 and 1604) are marked by green stars, and other 13 identified phosphorylation sites (S14, S414, S792, S818, S833, T838, T969, S1085, S1199, T1201, S1202, T1353, S1608) [40] are marked by black stars. The putative MAPK docking site is marked by a yellow line. (B) Localization of HAM-5-GFP to puncta localized to CAT tips during chemotropic interactions between genetically identical cells. HAM-5-GFP showed dynamic localization to CAT tips of germlings with an oscillation of every four min (arrow). HAM-5-GFP also localized to puncta within germlings and near nuclear compartments devoid of HAM-5-GFP (asterisks). The image left is a bright field image. Scale bar = 10 µM. (C) HAM-5-GFP localized to the sites of contact during germling fusion (arrow). (D) Western blots of WT, WT ( ham-5-gfp ) and Δ mak-2 ( ham-5-gfp ) germlings with immunoprecipitated HAM-5-GFP probed with anti-GFP antibodies (right panel shows longer run showing higher mobility of HAM-5-GFP in wild type germlings) specifically detecting HAM-5-GFP (210 kD; Figure S4D ). Lower panel shows a Western blot with identical samples probed with anti-phospho antibodies that specifically detect phosphorylated serine or threonine residues followed by a proline. (E) Localization of HAM-5-GFP to puncta in Δ mak-2 germlings. Some puncta showed localization to germling tips, but which did not oscillate during growth (white arrows). Scale bar = 10 µM.

    Article Snippet: Detection of phosphorylated HAM-5-GFP was performed using anti-phosphothreonine-proline/phosphoserine-proline antibodies (Abcam).

    Techniques: Western Blot, Immunoprecipitation

    The WD40 domain of HAM-5 interacts with MAK-2, while the C-terminus interacts with MEK-2. (A) Localization of HAM-5 1-351 -GFP (WD40 domain only) in WT germlings localized to the cytoplasm, the nucleus and the puncta at the cell periphery and at the tip during chemotropic interactions and oscillation (arrow). (B) HAM-5 1-351 -GFP in Δ ham-5 germlings localized to the cytoplasm and the nucleus; no puncta were observed. The images on the left are bright field images. Scale bar = 10 µM. (C) Western blots showing a specific interaction between MEK-2-mCherry (82.9 kD) with full length HAM-5-GFP (210 kD) or HAM-5 Δ67-348 -GFP (180 kD), but not with free GFP (27 kD; Figure S4C , D). An interaction between MEK-2-mCherry and HAM-5 1-351 -GFP (65.3 kD) was not detected. Input panels show Western blot of immunoprecipitated proteins isolated from 5 hr-old germlings probed with either anti-mCherry antibodies or anti-GFP antibodies. Output panel shows anti-mCherry immunoprecipitated proteins (MEK-2-mCherry) probed with anti-GFP antibodies (HAM-5-GFP). (D) Western blots showing a specific interaction between full length HAM-5-GFP or HAM-5 1-351 -GFP and phosphorylated MAK-2. Input panels show Western blot of proteins isolated from 5 hr-old germlings probed with anti-p42/44 antibodies (which recognize phosphorylated MAK-2 (40.8 kD; Figure S4D , E) [8] ) or immunoprecipitated proteins probed with anti-GFP antibodies. The output panel shows anti-GFP immunoprecipitated protein sample probed with anti-p42/44 antibodies, showing interaction between HAM-5-GFP or HAM-5 1-351 -GFP and phosphorylated MAK-2. (E) Schematic showing regions of interaction between HAM-5 and MAK-2 or MEK-2 based on co-immunoprecipitation experiments.

    Journal: PLoS Genetics

    Article Title: HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

    doi: 10.1371/journal.pgen.1004783

    Figure Lengend Snippet: The WD40 domain of HAM-5 interacts with MAK-2, while the C-terminus interacts with MEK-2. (A) Localization of HAM-5 1-351 -GFP (WD40 domain only) in WT germlings localized to the cytoplasm, the nucleus and the puncta at the cell periphery and at the tip during chemotropic interactions and oscillation (arrow). (B) HAM-5 1-351 -GFP in Δ ham-5 germlings localized to the cytoplasm and the nucleus; no puncta were observed. The images on the left are bright field images. Scale bar = 10 µM. (C) Western blots showing a specific interaction between MEK-2-mCherry (82.9 kD) with full length HAM-5-GFP (210 kD) or HAM-5 Δ67-348 -GFP (180 kD), but not with free GFP (27 kD; Figure S4C , D). An interaction between MEK-2-mCherry and HAM-5 1-351 -GFP (65.3 kD) was not detected. Input panels show Western blot of immunoprecipitated proteins isolated from 5 hr-old germlings probed with either anti-mCherry antibodies or anti-GFP antibodies. Output panel shows anti-mCherry immunoprecipitated proteins (MEK-2-mCherry) probed with anti-GFP antibodies (HAM-5-GFP). (D) Western blots showing a specific interaction between full length HAM-5-GFP or HAM-5 1-351 -GFP and phosphorylated MAK-2. Input panels show Western blot of proteins isolated from 5 hr-old germlings probed with anti-p42/44 antibodies (which recognize phosphorylated MAK-2 (40.8 kD; Figure S4D , E) [8] ) or immunoprecipitated proteins probed with anti-GFP antibodies. The output panel shows anti-GFP immunoprecipitated protein sample probed with anti-p42/44 antibodies, showing interaction between HAM-5-GFP or HAM-5 1-351 -GFP and phosphorylated MAK-2. (E) Schematic showing regions of interaction between HAM-5 and MAK-2 or MEK-2 based on co-immunoprecipitation experiments.

    Article Snippet: Detection of phosphorylated HAM-5-GFP was performed using anti-phosphothreonine-proline/phosphoserine-proline antibodies (Abcam).

    Techniques: Western Blot, Immunoprecipitation, Isolation

    Physical interaction of SP2 with the Cd14 promoter derived from C3Bir. A SP2 electrophoretic mobility shift was performed running a biotin-labelled Sp2 consensus oligonucleotide alone (lane 1) or incubated with a nuclear extracts isolated from RAW264.7 cells (lane 2). In the latter two complex formations (I and II) from the Sp2 consensus oligo with nuclear extract components were detected. Competition of increasing amounts of unlabelled SP2 oligo for binding to the nuclear extract proteins led to a stepwise disappearance of biotin-Sp2-oligo protein complexes (lane 3–5). After pre-incubation of the nuclear extracts with an SP2 specific antibody new complexes (III-V) were established in this supershift assay demonstrating physical interaction of the SP2 protein with its consensus oligo (lane 6). Specific SP2 DNA complex formation was also demonstrated by the failure of SP1 and SP3 antibodies to bind to the protein DNA complexes (lane 7 and 8). B BMMs after four days of culture. The scale bar represents 10μm at a 10x magnification. C The MMULV-mediated overexpression of mouse SP2 in primary BMMs derived from C3Bir mice correlated with a higher Cd14 transcription rate compared to untreated and control virus infected macrophages. ***: p

    Journal: PLoS ONE

    Article Title: Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir

    doi: 10.1371/journal.pone.0155821

    Figure Lengend Snippet: Physical interaction of SP2 with the Cd14 promoter derived from C3Bir. A SP2 electrophoretic mobility shift was performed running a biotin-labelled Sp2 consensus oligonucleotide alone (lane 1) or incubated with a nuclear extracts isolated from RAW264.7 cells (lane 2). In the latter two complex formations (I and II) from the Sp2 consensus oligo with nuclear extract components were detected. Competition of increasing amounts of unlabelled SP2 oligo for binding to the nuclear extract proteins led to a stepwise disappearance of biotin-Sp2-oligo protein complexes (lane 3–5). After pre-incubation of the nuclear extracts with an SP2 specific antibody new complexes (III-V) were established in this supershift assay demonstrating physical interaction of the SP2 protein with its consensus oligo (lane 6). Specific SP2 DNA complex formation was also demonstrated by the failure of SP1 and SP3 antibodies to bind to the protein DNA complexes (lane 7 and 8). B BMMs after four days of culture. The scale bar represents 10μm at a 10x magnification. C The MMULV-mediated overexpression of mouse SP2 in primary BMMs derived from C3Bir mice correlated with a higher Cd14 transcription rate compared to untreated and control virus infected macrophages. ***: p

    Article Snippet: Primary antibodies were applied to bind to CD14 (rat monoclonal [Sa14-2] to CD14 [FITC], Abcam, Cambridge, UK) or to beta actin (mouse monoclonal [mAbcam 8226] to beta Actin, Abcam).

    Techniques: Derivative Assay, Electrophoretic Mobility Shift Assay, Incubation, Isolation, Binding Assay, Over Expression, Mouse Assay, Infection

    Transfection system establishment. A Western blot analysis of CD14 expression in untreated and LPS stimulated murine RAW264.7 macrophages and NIH/3T3 fibroblasts, β-actin as endogeous control. Transfection efficiency test by determination of GFP-positive RAW264.7 cells by cyto fluorescence 24 hours after transfection with B Xtreme Gene HP, C Xtreme Gene 9 DNA, D Superfect, E Polyfect, and F by β-galactosidase assay, ***: p

    Journal: PLoS ONE

    Article Title: Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir

    doi: 10.1371/journal.pone.0155821

    Figure Lengend Snippet: Transfection system establishment. A Western blot analysis of CD14 expression in untreated and LPS stimulated murine RAW264.7 macrophages and NIH/3T3 fibroblasts, β-actin as endogeous control. Transfection efficiency test by determination of GFP-positive RAW264.7 cells by cyto fluorescence 24 hours after transfection with B Xtreme Gene HP, C Xtreme Gene 9 DNA, D Superfect, E Polyfect, and F by β-galactosidase assay, ***: p

    Article Snippet: Primary antibodies were applied to bind to CD14 (rat monoclonal [Sa14-2] to CD14 [FITC], Abcam, Cambridge, UK) or to beta actin (mouse monoclonal [mAbcam 8226] to beta Actin, Abcam).

    Techniques: Transfection, Western Blot, Expressing, Fluorescence

    Patterns of transcription factor binding sites associated with inflammation and colitis. Bioinformational analysis of the Cd14 alleles from the B6 and the C3Bir strain revealed striking changes in transcription factor binding sites caused by SNPs. AP1 and Sp1 sites were common in both alleles contributing to the control of Cd14 expression in mice; +1: translation start site; grey boxes: exons 1 and 2.

    Journal: PLoS ONE

    Article Title: Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir

    doi: 10.1371/journal.pone.0155821

    Figure Lengend Snippet: Patterns of transcription factor binding sites associated with inflammation and colitis. Bioinformational analysis of the Cd14 alleles from the B6 and the C3Bir strain revealed striking changes in transcription factor binding sites caused by SNPs. AP1 and Sp1 sites were common in both alleles contributing to the control of Cd14 expression in mice; +1: translation start site; grey boxes: exons 1 and 2.

    Article Snippet: Primary antibodies were applied to bind to CD14 (rat monoclonal [Sa14-2] to CD14 [FITC], Abcam, Cambridge, UK) or to beta actin (mouse monoclonal [mAbcam 8226] to beta Actin, Abcam).

    Techniques: Binding Assay, Expressing, Mouse Assay

    Truncations of the Cd14 promoter alleles. Constructs of the 5’ truncated Cd14 promoter were cloned in the pGL4.17 reporter plasmid and transfected in RAW264.7 cells. A The strong luciferase activity increase after removal of the distal 100 bp and of the sequence between -474 and -385 in the B6 allele indicated inhibitory elements in these regions. Activating binding motifs were identified through the drop of luciferase activity after cutting of the -844 to -722 and -298 to -181 regions. B Stepwise truncation of the C3Bir allele revealed significant expression peaks after removal of the -474 to -385 region and the -181 to -52 sequence while the -298 to -181 and the -52 to +85 regions seemed to contain stimulating binding motifs. ***: P

    Journal: PLoS ONE

    Article Title: Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir

    doi: 10.1371/journal.pone.0155821

    Figure Lengend Snippet: Truncations of the Cd14 promoter alleles. Constructs of the 5’ truncated Cd14 promoter were cloned in the pGL4.17 reporter plasmid and transfected in RAW264.7 cells. A The strong luciferase activity increase after removal of the distal 100 bp and of the sequence between -474 and -385 in the B6 allele indicated inhibitory elements in these regions. Activating binding motifs were identified through the drop of luciferase activity after cutting of the -844 to -722 and -298 to -181 regions. B Stepwise truncation of the C3Bir allele revealed significant expression peaks after removal of the -474 to -385 region and the -181 to -52 sequence while the -298 to -181 and the -52 to +85 regions seemed to contain stimulating binding motifs. ***: P

    Article Snippet: Primary antibodies were applied to bind to CD14 (rat monoclonal [Sa14-2] to CD14 [FITC], Abcam, Cambridge, UK) or to beta actin (mouse monoclonal [mAbcam 8226] to beta Actin, Abcam).

    Techniques: Construct, Clone Assay, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Sequencing, Binding Assay, Expressing

    Cd14 promoter activity after site-directed mutagenesis. A After inactivation of OCT1, Nrf2 and proximal AP1 binding sites in the B6 allele reduced activities were detected while in promoter constructs lacking Stat1/Cdx2 and SP1 led to complete transcriptional silencing. B In the C3Bir allele deletion of AP1 and SP2 binding sites resulted in significant transcriptional decrease, while after the mutation of SP1 only a slight reduction of Luciferase expression was seen. ***: P

    Journal: PLoS ONE

    Article Title: Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir

    doi: 10.1371/journal.pone.0155821

    Figure Lengend Snippet: Cd14 promoter activity after site-directed mutagenesis. A After inactivation of OCT1, Nrf2 and proximal AP1 binding sites in the B6 allele reduced activities were detected while in promoter constructs lacking Stat1/Cdx2 and SP1 led to complete transcriptional silencing. B In the C3Bir allele deletion of AP1 and SP2 binding sites resulted in significant transcriptional decrease, while after the mutation of SP1 only a slight reduction of Luciferase expression was seen. ***: P

    Article Snippet: Primary antibodies were applied to bind to CD14 (rat monoclonal [Sa14-2] to CD14 [FITC], Abcam, Cambridge, UK) or to beta actin (mouse monoclonal [mAbcam 8226] to beta Actin, Abcam).

    Techniques: Activity Assay, Mutagenesis, Binding Assay, Construct, Luciferase, Expressing

    Expression of TLR9 on the surface of human neutrophils stimulated with Hp-DNA and H. pylori . Neutrophils (2.5×10 5 ) were incubated in the presence of Hp-DNA (1 µg/mL) or H. pylori (2.5×10 7 bacteria) or unstimulated (control) for 3 h and the TLR9 expression were analyzed by confocal microscopy ( A ) and by flow cytometry ( B–D ). For confocal microscopy, neutrophils were stained with fluorescein isothiocyanate-conjugated anti-TLR9 Ab (green) and nuclei stained with TOTO-3 iodide (red); and for flow cytometry they were staining with FITC-conjugated anti-TLR9 Ab (purple, red and green histograms); and isotype control (blue histogram) and unstained neutrophils were included (black line), the MFI data is shown in B . This figure represents one of three ( A ) and six ( B ) independent experiments performed. The MFI and positive cells in % data are represented as mean + SD of six independent experiments ( C and D respectively).

    Journal: PLoS ONE

    Article Title: TLR9 and NF-?B Are Partially Involved in Activation of Human Neutrophils by Helicobacter pylori and Its Purified DNA

    doi: 10.1371/journal.pone.0101342

    Figure Lengend Snippet: Expression of TLR9 on the surface of human neutrophils stimulated with Hp-DNA and H. pylori . Neutrophils (2.5×10 5 ) were incubated in the presence of Hp-DNA (1 µg/mL) or H. pylori (2.5×10 7 bacteria) or unstimulated (control) for 3 h and the TLR9 expression were analyzed by confocal microscopy ( A ) and by flow cytometry ( B–D ). For confocal microscopy, neutrophils were stained with fluorescein isothiocyanate-conjugated anti-TLR9 Ab (green) and nuclei stained with TOTO-3 iodide (red); and for flow cytometry they were staining with FITC-conjugated anti-TLR9 Ab (purple, red and green histograms); and isotype control (blue histogram) and unstained neutrophils were included (black line), the MFI data is shown in B . This figure represents one of three ( A ) and six ( B ) independent experiments performed. The MFI and positive cells in % data are represented as mean + SD of six independent experiments ( C and D respectively).

    Article Snippet: To flow cytometry, treated neutrophils were resuspended in blocking buffer (PBS containing 2% FBS, 2% rabbit serum, 5 mM EDTA and 0.1% sodium azide) and incubated on ice for 1 h. The cells suspension was centrifuged and stained with fluorescein isothiocyanate-conjugated anti-TLR9 Ab (Abcam, Cambridge, UK).

    Techniques: Expressing, Incubation, Confocal Microscopy, Flow Cytometry, Cytometry, Staining