Journal: Physiological Reports

Article Title: Decreased stability of erythroblastic islands in integrin β3-deficient mice

doi: 10.1002/phy2.18

Figure Lengend Snippet: Assessment of local fibronectin expression in bone marrow. (A) Examples of immunofluorescence against fibronectin (original magnification 200×). (B) Illustration of the quantification procedure. Using an image analyzing system a threshold 20% above the average background optical density level (oDL) was defined. A circular sample tool was used to measure each single vessel in the images separately. The threshold procedure defined the pixels representing each single vessel (red). Then the average oDL of each vessel was calculated. (C) These measurements were very stable and revealed in bone marrow vessels an increased fibronectin staining intensity in β3 −/− mice whereas it was reduced in tg6 mice. Between both wt control groups no differences could be detected ( n = 4).

Article Snippet: Other bone marrow sections were immunostained for fibronectin (rabbit anti-human fibronectin, ICN/Cappel; goat anti-rabbit IgG, DyLight 649 labeled, Jackson ImmunoResearch Lab., Rheinfelden, Switzerland), cover slipped using a moviol-based (Calbiochem, Zug, Switzerland) embedding medium (Osborn and Weber ) containing 1,4-Diazobicyclo-[2.2.2]-Octan (Sigma) and hardened overnight.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 1. Flow cytometry analyses. A, Example of gating strategy to analyze the binding of scFv (single-chain fragment variable)-phages on rabbit atheromatous proteins coupled to beads. The control (mouse anti-pVIII and Alexa Fluor 488–conjugated anti-mouse antibodies) and the P3 clone are illustrated. Binding of scFv-phages (B) and soluble scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) (C) on rabbit atheromatous proteins coupled to beads. Protein binding was detected using a mouse anti-pVIII antibody and an Alexa Fluor 488-conjugated anti-mouse antibody for scFv-phages, and using a rabbit anti-human Fc antibody and an Alexa Fluor 488–conjugated anti-rabbit antibody for scFv-Fc. Mean values±SD were calculated using the geometric mean of fluorescence (P2 geomean) obtained with 3 different antibody batches. The error bars represent the mean values±SD. CN indicates control negative; FSC-A,forward scatter area; VH, variable heavy; VL, variable light domains.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Flow Cytometry, Binding Assay, Control, Protein Binding, Fluorescence

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 2. Immunohistochemical analysis of arterial tissue sections from atheromatous rabbits and Apoe−/− mice and human endarterectomy specimens using the indicated scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G). The different areas in transversal sections are identified: adventitia (A), media (M), and intima (I). Sections were incubated with the indicated scFv-Fc antibodies (P3, C4, D4, and C8), followed by the HRP-conjugated goat anti-human Fcγ antibody, and the DAB substrate kit reagent. The yellow-brown staining indicates the presence of the antigen recognized by the scFv-Fc. No staining was observed in mouse and rabbit sections incubated with the secondary antibody alone, and there is low background noise in the human sections (upper panels). Scale bars, 100 µm. Nuclei were counterstained with hematoxylin. DAB indicates 3,3′-diaminobenzidine; and HRP, horseradish peroxidase.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Immunohistochemical staining, Incubation, Staining

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 3. SDS-PAGE analysis of immune complexes. After immunoprecipitate elution in 2×SDS Laemmli buffer, immune complexes were separated on polyacrylamide gels (4%–10%) and stained with silver nitrate. SDS-PAGE 1 and 2: proteins extracted from human endarterectomy specimens were immunoprecipitated with the P3 and C4 scFv-Fc (single- chain fragment variable fused to the crystallizable fragment of immunoglobulin G) (from mammalian cells), respectively. SDS-PAGE 3: Gal3R (recombinant galectin-3 protein) was immunoprecipitated with a commercial anti-GAL3 Ab (anti-galectin-3 antibody). MW indicates molecular weight; M, marker; NA, non attributed; PH, proteins from human biopsies.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: SDS Page, Staining, Immunoprecipitation, Recombinant, Molecular Weight, Marker

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 4. Binding of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) and control antibodies to recombinant proteins by ELISA assay. A, P3 scFv-Fc showed stronger binding to recombinant galectin-3 than to the galectin-3BP (galectin-3 binding protein) ligand. Galectin-1 and Glycoprotein αIIbβ3 (GPαIIbβ3) were used to assess the specificity of galectin-3 binding. An irrelevant antigen (BSA) served as a negative control. P3 scFv-Fc binding was detected with a horseradish peroxidase (HRP)-conjugated anti-human Fcγ antibody, and absorbance was measured at 405 nm. Data are the means of 3 independent experiments repeated in triplicate. According to the Kruskal-Wallis test followed by the Dunn test, differential analyses provided the following P values: P=0.0222 for galectin-3 vs galectin-3BP, P=0.0020 for galectin-3 vs galectin-1, P=0.0178 for galectin-3 vs BSA, and P=0.1087 for galectin-3 vs GPαIIbβ3. B, The specificity of the commercial antibodies against galectin-3, galectin-1, and galectin-3BP, of the mouse AP2 (AP2 clone) antibody (from Dr Nurden) against GPαIIbβ3 integrin, and of CN (control negative) scFv-Fc (negative control; from Laboratoire Français de Fractionnement et de Biotechnologies) was assesses by ELISA with recombinant galectin-3, galectin-1, galectin-3BP, BSA, and GPαIIbβ3. The anti-galectin-3 and anti-galectin-3BP antibodies were detected with an HRP-conjugated anti-rabbit antibody. The galectin-1 and AP2 antibodies were detected with an HRP-conjugated anti-mouse antibody. CN was detected with a peroxidase- conjugated anti-human FCγ antibody. The error bars reprement the mean values±SD.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Binding Assay, Control, Recombinant, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 5. Colocalization of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) with an anti-galectin-3 antibody and an anti-LOX1 (lectin-type oxidized LDL receptor 1) antibody in the intima of human endarterectomy specimens and coronary sections. A through D, Co-staining of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) and an anti-galectin-3 antibody in human endarterectomy sections by immunofluorescence. An Alexa Fluor 488 anti-mouse antibody (+488 anti-Ms [mouse]) was used to reveal the specific binding of the commercial anti-human galectin-3 antibody (Ms anti-galectin-3) (A and C). The Alexa Fluor 568 anti-human antibody (+568 anti-Hum) was used to reveal the specific binding of P3 scFv-Fc (Hum P3 scFv-Fc) (B and C). Before image merging, the red and green fluorescence signals were adjusted to comparable levels. The yellow color indicates colocalization of the antigens recognized by P3 scFv-Fc and the anti-human galectin-3 antibody (C). Secondary antibodies alone were used as negative controls (D). Size bars: 100 µm. E through H, The Alexa Fluor 488 anti-rabbit antibody (+488 anti-Rb [rabbit]) was used to reveal the specific binding of the commercial anti-LOX1 antibody (Rb anti-LOX1) (E and G). The Alexa Fluor 647 anti-human antibody (+568 anti-Hum) was used to reveal the specific binding of P3-scFv-Fc (Hum P3 scFv-Fc) (F and G). Before image merging, the red and green fluorescence signals were adjusted to comparable levels. The yellow color indicates colocalization of the antigens labeled by P3 scFV-Fc and anti-LOX1 antibody (G). As galectin-3 is also expressed also by other cell types (ie, mast cells, eosinophils, neutrophils, endothelial cells, and activated T and B cells8,27), areas outside the macrophage location also are stained by P3 scFv-Fc (G). Size bars: 100 μm.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Staining, Immunofluorescence, Binding Assay, Fluorescence, Labeling

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 6. In vitro immunofluorescence analysis of Apoe−/− aorta sections with P3 scFv-Fc (single- chain fragment variable fused to the crystallizable fragment of immunoglobulin G) and an anti- LOX1 (lectin-type oxidized LDL receptor 1) antibody. Colocalization analysis by fluorescence microscopy in Apoe−/− mouse aorta sections of the antigens recognized by P3 scFv-Fc and the commercial anti-LOX1 antibody. The Alexa Fluor 488 anti-rabbit antibody (+488 anti-Rb) was used to reveal the specific binding of the anti-LOX1 antibody (Rb anti-LOX1). The Alexa Fluor 647 anti-human antibody (+647 anti-Hum) was used to reveal the specific binding of P3 scFv-Fc (Hum P3 scFv-Fc). Before image merging, the red and green fluorescence signals were adjusted to comparable levels. The yellow color and yellow arrowheads indicate colocalization of the antigens recognized by P3 scFv-Fc and the anti-LOX1 antibody. Size bars: 250 μm.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: In Vitro, Immunofluorescence, Fluorescence, Microscopy, Binding Assay

Journal: Journal of the American Heart Association

Article Title: In Vivo Human Single‐Chain Fragment Variable Phage Display‐Assisted Identification of Galectin‐3 as a New Biomarker of Atherosclerosis

doi: 10.1161/jaha.120.016287

Figure Lengend Snippet: Figure 7. Ex vivo imaging of P3 scFv-Fc (single-chain fragment variable fused to the crystallizable fragment of immunoglobulin G) in Apoe−/− and wild-type (WT) mice using a fluorescent ultramicroscope. Fluorescence macroscopy analysis of P3 scFv-Fc coupled to Alexa Fluor 568 dye after ex vivo injection in Apoe−/− and WT mice. A human immunoglobulin (Ig)G coupled to Alexa Fluor 568 was used as negative control antibody. P3 scFv-Fc shows specific labeling of the atheroma in the Apoe−/− mouse (yellow arrowheads). Size bars: 500 μm.

Article Snippet: This was followed by incubation with mouse antipVIII (Abcam, France) or rabbit anti- human Fcγ (Jackson Immunoresearch, USA) primary antibodies (1:1000) at 4°C under rotation overnight, and by incubation with Alexa Fluor 488– labeled anti- mouse antibodies (Life Technologies, France) (1:40) for scFv- phage detection.

Techniques: Ex Vivo, Imaging, Fluorescence, Injection, Negative Control, Labeling

Journal: Pathogens

Article Title: Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells

doi: 10.3390/pathogens8040259

Figure Lengend Snippet: Pseudovirus production after co-transfection of three plasmids in human embryonic kidney (HEK)-293T cells at 72 h post transfection. ( a ) Production of red fluorescence protein (RFP)-bound green fluorescence protein (GFP)-fused S protein of Scotophilus bat coronavirus (CoV)-512 pseudovirus (RFP-Sco-S-eGFP): pictures from left to right are HEK-293T cells under bright field, red fluorescence produced by the expressed red fluorescence protein (RFP) from the transfer plasmid pLAS2w.RFP-C.Pneo, green fluorescence produced by the expressed green fluorescence protein (GFP) fused with the carboxyl terminal spike (S) protein of Scotophilus bat CoV-512. ( b ) Production of severe acute respiratory syndrome (SARS)-CoV (RFP-SARS-S): HEK-293T cells on the left and red fluorescence from RFP on the right. ( c ) Production of vesicular stomatitis virus (VSV) pseudovirus (RFP-VSV-G): HEK-293T cells on the left and red fluorescence from RFP on the right. The scale bars represent 100 µm.

Article Snippet: To detect the GFP fused to S protein of Scotophilus bat CoV-512 pseudovirus, the mAb to GFP (Thermo Fisher in 1:200 was used as primary antibody and 1:100 Alexa Fluor ® 594 AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as secondary antibody to distinguish the green fluorescence from GFP and red fluorescence from bound secondary antibodies.

Techniques: Cotransfection, Transfection, Fluorescence, Produced, Plasmid Preparation, Virus

Journal: Pathogens

Article Title: Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells

doi: 10.3390/pathogens8040259

Figure Lengend Snippet: Pseudovirus titration from the dilutions of ×1 to ×10 −4 into HEK-293T cells. ( a ) Infection of Scotophilus bat coronavirus-512 pseudovirus (RFP-Sco-S-eGFP) was indicated by the expression of red fluorescent protein (RFP) in cells on top panel and by the expression of green fluorescent protein (GFP) fused to the spike (S) protein on bottom panel. ( b ) Infection of severe acute respiratory syndrome coronavirus pseudovirus (RFP-SARS-S) was indicated by the expression of RFP. ( c ) Infection of vesicular stomatitis virus pseudovirus (RFP-VSV-G) was indicated by the expression of RFP. The scale bars represent 100 µm.

Article Snippet: To detect the GFP fused to S protein of Scotophilus bat CoV-512 pseudovirus, the mAb to GFP (Thermo Fisher in 1:200 was used as primary antibody and 1:100 Alexa Fluor ® 594 AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as secondary antibody to distinguish the green fluorescence from GFP and red fluorescence from bound secondary antibodies.

Techniques: Titration, Infection, Expressing, Virus

Journal: Pathogens

Article Title: Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells

doi: 10.3390/pathogens8040259

Figure Lengend Snippet: Detection of p24 protein in the lentiviral backbone of pseudoviruses and green fluorescence protein (GFP) fused with the spike protein of Scotophilus bat coronavirus-512 pseudovirus (RFP-Sco-S-eGFP) by immunofluorescence antibody (IFA) assay. ( a ) Positive green IFA signals for p24 in HEK-293T cells infected with vascular stomatitis virus pseudovirus (RFP-VSV-G). ( b ) Positive green IFA signals for p24 in HEK-293T cells infected with severe acute respiratory syndrome coronavirus pseudovirus (RFP-SARS-S). ( c ) Positive green IFA signals for p24 in HEK-293T cells infected with RFP-Sco-S-eGFP. ( d ) Positive green IFA signals for GFP in HEK-293T cells infected with RFP-Sco-S-eGFP. ( e ) Positive green IFA signals for p24 in MFK cells infected with FLuc-VSV-G. ( f ) Positive green IFA signals for p24 in MFK cells infected with FLuc-SARS-S. ( g ) Positive red IFA signals and green fluorescence from GFP in MFK cells infected with FLuc-Sco-S-eGFP. ( h ) Positive red IFA signals and green fluorescence from GFP in MFK cells infected with FLuc-Sco-S-eGFP. Blue parts are DAPI (4′,6-diamidino-2-phenylindole) staining cell nucleus. The scale bars represent 100 µm.

Article Snippet: To detect the GFP fused to S protein of Scotophilus bat CoV-512 pseudovirus, the mAb to GFP (Thermo Fisher in 1:200 was used as primary antibody and 1:100 Alexa Fluor ® 594 AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as secondary antibody to distinguish the green fluorescence from GFP and red fluorescence from bound secondary antibodies.

Techniques: Fluorescence, Immunofluorescence, Infection, Virus, Staining

Journal: Pathogens

Article Title: Entry of Scotophilus Bat Coronavirus-512 and Severe Acute Respiratory Syndrome Coronavirus in Human and Multiple Animal Cells

doi: 10.3390/pathogens8040259

Figure Lengend Snippet: Cell entries of pseudoviruses carrying red fluorescence protein (RFP) gene are represented by red fluorescence images (RFP) for severe acute respiratory syndrome coronavirus pseudovirus (RFP-SARS-S, middle panels) and vesicular stomatitis virus pseudovirus (RFP-VSV-G, right panels), and merged red and green fluorescence images (RFP and GFP) present the entries of Scotophilus bat coronavirus-512 pseudovirus (RFP-Sco-S-eGFP, left panels) in 11 different cells. ( a ) HEK-293T: human ( Homo sapiens ) embryonic kidney epithelial cells; ( b ) Caco-2: human ( Homo sapiens ) colorectal adenocarcinoma cells; ( c ) Vero: African green monkey ( Chlorocebus aethiops ) kidney epithelial cells; ( d ) IEC-6: rat ( Rattus norvegicus ) small intestine epithelial cells; ( e ) PK15: pig ( Sus scrofa ) kidney epithelial cells; ( f ) MDCK: Madin Darby dog ( Canis familiaris ) kidney epithelial cells; ( g ) Fcwf-4: cat ( Felis catus ) whole fetus cells; ( h ) Pabr: black flying fox (Pteropus alecto) brain cells; ( i ) Palu: black flying fox ( Pteropus alecto ) lung epithelial cells; ( j ) Paki: black flying fox ( Pteropus alecto ) kidney epithelial cells; ( k ) MFK: Eastern bent-winged bat ( Miniopterus fuliginosus ) kidney epithelial cells. Bright field images of cells were used as reference. The scale bars represent 100 µm.

Article Snippet: To detect the GFP fused to S protein of Scotophilus bat CoV-512 pseudovirus, the mAb to GFP (Thermo Fisher in 1:200 was used as primary antibody and 1:100 Alexa Fluor ® 594 AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as secondary antibody to distinguish the green fluorescence from GFP and red fluorescence from bound secondary antibodies.

Techniques: Fluorescence, Virus