fluorescein antibody Abcam Search Results


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  • 99
    Thermo Fisher dapi
    PNx induces increased nuclei numbers in α 1 +/− mice. Left ventricle sections from each experimental group were stained with <t>4′,6-diamidino-2-phenylindole</t> <t>(DAPI;</t> blue) and cardiac <t>troponin</t> I (cTnI, red) as described in materials
    Dapi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 155806 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore gfp
    IKKβ and IKKε phosphorylate YAP. ( a ) 293T cells were co-transfected with <t>GFP-YAP</t> and Flag-IKKα, HA-IKKβ or IKKε. The cell lysates were analyzed by western blot with indicated antibodies. The asterisk showed the shifted band ( b , c ) 293T cells were co-transfected with GFP-YAP and HA-IKKβ or IKKε. Cell lysates were immunoprecipitated with indicated antibodies and analyzed by western blot. ( d ) 293T cells were transfected with GFP-YAP and HA-IKKβ or IKKε. The cell lysates were analyzed by phos-tag gel western blot. The asterisk showed the phosphorylated band. ( e ) The recombinant GST-YAP protein was incubated with immunoprecipitated HA-IKKβ or IKKε in phosphorylation buffer. Reactions were subjected to electrophoresis and immunoblotted with antibody against pan-phosphorylated serine/threonine (S/T). ( f ) MCF7 cells were treated with TNFα for indicated times, the cells were then lysed for phos-tag gel western blot. The asterisk showed the phosphorylated band. ( g ) 293T cells were transfected with GFP-YAP and HA-IKKβ/ε. Cell lysates were immunoprecipitated with GFP antibody followed by immunoblotting with <t>TEAD4</t> antibody. ( h ) MCF7 cells were treated with 10 μ M TPCA-1 or 10 μ M IKK-16 for 12 h. Then endogenous YAP was immunoprecipitated and followed by immunoblotting with TEAD4 antibody.
    Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (Abcam)
    99
    Abcam gfp
    Specialization of Sema3a + cardiomyocytes into the conduction system in the developing heart. ( a – f ) Z-stack images of <t>RFP</t> and <t>GFP</t> immunostaining on Sema3a-CreERT2; R26-tdTomato; Cx40-GFP heart sections. Tamoxifen was administered at E12.5 ( a , b ), E14.5 ( c , d ) and E18.5 ( e , f ). The hearts were collected at P7 and P21 for each group. YZ indicates signals from the dotted lines on the Z-stack images. Scale bars, 50 µm. Each image is representative of 5 individual samples. ( g ) Schematic figure showing Sema3a + cells (red) and Cx40 + cells (green) in the developing and adult heart.
    Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 8593 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hoechst 33342
    Dox-dependent TS expression in TFTS66 cells. A.  TS antigens in cell lysates of TFTS66 transformant were detected by immunoblotting using anti-human TS mouse monoclonal antibody. The standard cell lysates (0 ng/ml Dox) were titrated, and a standard curve for detection was obtained from the signal intensity on the digitized image (upper panel). Using the highly linear detection characteristics (p = 0.997), TS expression levels were quantified: rectangle, TFTS66; circle, the control transformant, TFC7.  B.  TS expression in TFTS66 cells exposed to various concentrations of Dox was assessed by immunoblotting and similarly quantified. The symbols are shaded according to the Dox concentrations. Some of the data points are also shown in Fig 2A.  C.  The quantity of TS protein (TS total , see   Materials and methods ) and its catalytic activity were enzymatically assayed in lysates prepared from TFTS66 cells exposed to 0, 0.5 and 1.0 ng/ml Dox. The results are plotted as a function of the TS expression level determined by immunoblotting: open rectangle, Dox0; shaded rectangle, Dox0.5; closed rectangle, Dox1.0.  D.  TS expression in TFTS66 cells was observed using fluorescent immunocytochemistry. Cells grown on chamber slides were fixed and reacted with TS-specific antibody. Cellular distribution of TS antigens was visualized by red fluorescent signals. Cells were also counterstained with Hoechst 33342. Results obtained in TFTS66 (0, 0.1 and 1.0 ng/ml Dox) and its parental line, DLD-1, are shown (magnification X100).
    Hoechst 33342, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gfp tag polyclonal antibody
    AnkB and AnkG interact with NF155 in vivo and can be targeted to paranodes independently of paranodal junctions and NF155. ( a–d ) P8 sciatic nerve sections were stained for AnkB (N105/17), NFasc and Caspr ( a, b ), and the respective longitudinal line scans are shown ( c, d ). ( e ) Immunoprecipitation of AnkB from adult rat sciatic nerves co-precipitated NF155. Immunoprecipitation with the <t>anti-GFP</t> antibody served as a negative control. Heavy chain, mouse IgG heavy chain. AnkB was detected by H-300 rabbit <t>polyclonal</t> antibodies. ( f ) Immunoprecipitation of AnkG from P21 mouse spinal cords co-precipitated NF186 and NF155. AnkG was detected by the goat polyclonal antibodies. The immunoprecipitation in ( e, f ) was reproduced at least three times. The full blots are presented in Supplementary Fig. 3 . ( g–i ) Immunostaining of P7 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the residual AnkB and NFasc at paranodes. ( j–l ) Immunostaining of P7 spinal cords (AnkG, N106/36). Arrows point to the residual AnkG and NFasc at paranodes. ( m–o ) Immunostaining of P5 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the paranodes with residual AnkB. ( p–r ) Immunostaining of P12 Nfasc-cHet ( Cnp-Cre;Nfasc f/+ ) and Nfasc-cKO ( Cnp-Cre;Nfasc f/f ) spinal cords (AnkG, N106/36). Two mice per genotype and more than 100 nodes were examined in each mouse. 80–190 nodes per animal were quantified. Scale bars = ( a, b ) 5 μm for (a) and 3.3 μm for (b); 5 μm ( g–i , j–l , m–o , and p–r ).
    Gfp Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher gfp
    Molecular characterization of the pocket. pH Smo cilia ( green ) were fixed and first labeled with <t>anti-GFP</t> ( red ) in unpermeabilized cells to label the extracellular region ( arrowhead ) and then fixed again, permeabilized and stained with various ciliary markers ( blue ). a Acetylated <t>tubulin</t> (Ac Tub) stains the entire length of the ciliary axoneme and does not correlate with anti-GFP staining. b The anti-GFP staining ends above the basal body (BB), marked by pericentrin. c The anti-GFP staining also ends above the transition zone (TZ), marked by CEP290. d – f The anti-GFP staining inversely coincides with: EHD1 ( d ), Septin 9 ( e ) and glutamylated tubulin (Glu Tub) ( f )
    Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 24758 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology gfp
    BC transplantation rescued mouse pulmonary function . (A) Injured mouse lung without or with <t>GFP-labeled</t> SOX9 + BCs transplantation by anti-GFP and <t>anti-Fibronectin</t> co-staining. Scale bar, 200 μm. (B) Left, immunofluorescence image of injured mouse lung transplanted with GFP-labeled SOX9 + BCs; right, immunostaining on the same section showing exclusion of α-SMA+ myofibroblasts from GFP + area. Scale bar, 200 μm. (C) CO 2 partial pressure of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse. (D) O 2 partial pressure of mouse arterial blood 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse. (E) O 2 saturation of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse
    Gfp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 6556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam gfp antibody
    Degradation of Os WRKY11 protein by the UPS. a Rice ( Oryza sativa ) protoplasts were transformed with 35S:: OsWRKY11-YFP . After 4 h, protoplasts were treated with 100 μM MG132 and buffer for 1 day. Fluorescence was observed using a confocal laser scanning microscope. Scale bars: 30 μm. NT: non-treated control. b <t>Immunoblot</t> analysis of total protoplast proteins in the absence (−) or presence (+) of 100 μM MG132. Blots were probed with green fluorescent protein <t>(GFP)</t> antibody. The PAT antibody was used as a control for transformation efficiency
    Gfp Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1781 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    fitc  (Abcam)
    94
    Abcam fitc
    Membrane TK1 expression in of colon, breast, and lung cancer cell lines. Flow cytometry analysis of cell lines treated with anti-TK1 antibodies. a Quantification of TK1 expression on the cell membrane of HT-29 and SW620 cell lines stained with <t>FITC</t> or APC-conjugated anti-TK1 antibodies. b Quantification of TK1 expression on the cell membrane of MCF7 and MDA-MB-231 cell lines. The top bar graph shows MCF7 and MDA-MB-231 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. c Quantification of TK1 expression on the cell membrane of NCI-H460 and A549 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. Statistical analysis was performed by comparing the mouse isotype control fluorescent levels to those of A72, A74, CB1, or <t>ab91651.</t> *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001; ns = P > 0.05
    Fitc, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 1845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc gfp
    Subcellular localization of K3 is not different from wildtype <t>AID.</t> (A) Schematic representation of <t>AID-GFP</t> fusion constructs used for localization assays. GFP is fused to the C-terminus of AID or mutant K3. (B) HEK293T cells were transiently transfected with the respective GFP-fusion constructs. Two days post transfection, cells were treated as indicated with Leptomycin B (LMB) for 3 hours. Cells were fixed and localization of the respective GFP fusion proteins visualized using fluorescence microscopy. (C) The k46 B cell line stably expressing AID-GFP or K3-GFP fusion proteins were treated with or without Leptomycin B (LMB) for 3 hours and localization of GFP fusion constructs was determined using fluorescence microscopy.
    Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher fitc
    Hematopoietic cells in human placenta. Multiple clusters of CD34+cells, not associated with fetal or maternal circulation, are present in placental tissue. Significant numbers of CD34 cells are also positive for hematopoietic precursor markers such as CD90, CD38 and CD133. Paraffin sections of human placenta stained for CD34, CD90, CD38, and CD133 are shown. A1 : CD90 (green), A2 : CD34 (red), A3 : merged image of A1 and A2 with nuclear co-staining (DAPI blue), size indication bar is 10 µm. The white arrow points to CD34+CD90+cells. B1 : CD38 (green), B2 : CD34 (red), B3 : merged image of B1 and B2 with nuclear co-staining (DAPI blue), size indication bar is 100 µm. The white arrows point to CD34+ CD38+ cells. The green arrow points at a CD34+ capillary. C1 : CD133 (green), C2 : CD34 (red), C3 : merged image, showing staining for CD34 (red), CD133 (green), and nuclei (DAPI blue), size indication bar is 20 µm. The white arrows point to CD34+CD133+cells not associated with structures of the circulation (capillary walls). The green arrow points at the CD34+CD133+capillary. D : Negative control staining. D1 : merged image of staining with isotype anti-mouse antibody and isotype anti-rabbit antibody, secondary <t>FITC</t> (green) anti-mouse and <t>Alexa</t> Fluor 633 anti-rabbit antibody (red), co-stained for nuclei with DAPI (blue). No signals from cell markers other than nuclei are present. D2 : Merged image of staining with mouse anti-CD34 antibody and isotype anti-rabbit antibody, secondary FITC anti-mouse (green) and Alexa Fluor 633 anti-rabbit (red) antibody, co-stained for nuclei with DAPI (blue). Only signals from the CD34 cell marker and nuclei are present. D3 : Isotype anti-mouse antibody and anti-CD133 rabbit antibody, secondary FITC anti-mouse and Alexa Fluor 633 anti-rabbit antibody, co-staining DAPI (blue), merged image. Only a signal for CD133+cells is observed, no signals from CD34 cell markers are present. The size indication bar is 50 µm.
    Fitc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11713 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam antibodies against gfp
    β-catenin and TCF show enrichment when localized to the nucleus in successive cell cycles. A) Nuclear localization of Venus::SYS-1 in the ABpl lineage, shows the highest level in cells derived from consecutive posterior parents. B) Detail showing ABplaaa lineage; Black arrows show comparison between posterior cousin cells with high nuclear Venus::SYS-1 in two consecutive divisions (High-High or HH cells, ABplaaaapp marked “pp”), which have higher levels of nuclear Venus::SYS-1 than their cousins with parents that had no/low nuclear Venus::SYS-1 (Low-High or LH cells, ABplaaaaap marked “ap). C) Nuclear localization of <t>GFP::POP-1,</t> detectable after the 50 cell stage. GFP::POP-1 polarity is the opposite of Venus::SYS-1 with highest expression in cells derived from consecutive anterior parents. D) Detail of ABplppp lineage for GFP::POP-1 showing comparison between anterior cousin cells with high nuclear POP-1 in consecutive divisions (ABplpppaa marked “aa”) and their cousins with parents that had low nuclear POP-1 (ABplppppa marked “pa”). E) Venus::SYS-1 concentration for embryonic High-High cells (y axis) compared with their Low-High cousin cell. Most points fall above the red y = x line. F) Average GFP::POP-1 concentration for all High-High cells (y-axis) born after the 50 cell stage compared with their Low-High cousin cells. Most points fall above the red y = x line. G) Diagram illustrating comparison of SYS-1 and POP-1 asymmetry levels in daughter cells and graph showing POP-1 and SYS-1 asymmetry levels for all divisions after the 50-cell stage. Divisions of parent cells with high levels of POP-1 (blue) tend to have greater POP-1 asymmetry (x-axis) and less SYS-1 asymmetry (y-axis) than parent cells with high levels of SYS-1(red). H) Low/High labeling scheme and graph showing comparison of concentrations of Venus::SYS-1 and GFP::POP-1 for all individual cells after the 50-cell stage, colored by whether they are POP-1 High-High (dark blue), POP-1 Low-High (light blue), SYS-1 High-High (red) or SYS-1 Low-High (pink). The relative ratio of Venus::SYS-1 fluorescence intensity to GFP::POP-1 intensity for each group can be estimated from slope of a best-fit line (indicated above each line). Concentration values can be negative because nuclear intensity is subtracted from local background.
    Antibodies Against Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsa  (Abcam)
    99
    Abcam bsa
    β-catenin and TCF show enrichment when localized to the nucleus in successive cell cycles. A) Nuclear localization of Venus::SYS-1 in the ABpl lineage, shows the highest level in cells derived from consecutive posterior parents. B) Detail showing ABplaaa lineage; Black arrows show comparison between posterior cousin cells with high nuclear Venus::SYS-1 in two consecutive divisions (High-High or HH cells, ABplaaaapp marked “pp”), which have higher levels of nuclear Venus::SYS-1 than their cousins with parents that had no/low nuclear Venus::SYS-1 (Low-High or LH cells, ABplaaaaap marked “ap). C) Nuclear localization of <t>GFP::POP-1,</t> detectable after the 50 cell stage. GFP::POP-1 polarity is the opposite of Venus::SYS-1 with highest expression in cells derived from consecutive anterior parents. D) Detail of ABplppp lineage for GFP::POP-1 showing comparison between anterior cousin cells with high nuclear POP-1 in consecutive divisions (ABplpppaa marked “aa”) and their cousins with parents that had low nuclear POP-1 (ABplppppa marked “pa”). E) Venus::SYS-1 concentration for embryonic High-High cells (y axis) compared with their Low-High cousin cell. Most points fall above the red y = x line. F) Average GFP::POP-1 concentration for all High-High cells (y-axis) born after the 50 cell stage compared with their Low-High cousin cells. Most points fall above the red y = x line. G) Diagram illustrating comparison of SYS-1 and POP-1 asymmetry levels in daughter cells and graph showing POP-1 and SYS-1 asymmetry levels for all divisions after the 50-cell stage. Divisions of parent cells with high levels of POP-1 (blue) tend to have greater POP-1 asymmetry (x-axis) and less SYS-1 asymmetry (y-axis) than parent cells with high levels of SYS-1(red). H) Low/High labeling scheme and graph showing comparison of concentrations of Venus::SYS-1 and GFP::POP-1 for all individual cells after the 50-cell stage, colored by whether they are POP-1 High-High (dark blue), POP-1 Low-High (light blue), SYS-1 High-High (red) or SYS-1 Low-High (pink). The relative ratio of Venus::SYS-1 fluorescence intensity to GFP::POP-1 intensity for each group can be estimated from slope of a best-fit line (indicated above each line). Concentration values can be negative because nuclear intensity is subtracted from local background.
    Bsa, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 11355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fitc
    Effect of marker nucleotide, the detection approach and dTTP on the signal intensity. The samples containing Lep cells accidentally infected with mycoplasma were fixed by formaldehyde, permeabilized by Triton X-100 and mycoplasmas’ DNA was detected using enzymatic detection with Pol I. ( a ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). ( b ) The enzymatic mixture contained fluorescein-dUTP. ( c ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized by <t>streptavidin</t> conjugated with <t>FITC.</t> ( d ) The enzymatic mixture contained biotin-dUTP and an equimolar amount of dTTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). The images were acquired for 11.44 ms. Scale bar = 10 µm.
    Fitc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8829 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    AvesLabs gfp
    Coincidence of subpleural lymphatic plexuses and leakage sites. Image orientation: caudal (left), rostral (right). ADN–VEGF-C mice were analyzed after 0.075 mg/mL doxycycline for 7 days. A: Left panel: Whole mount of dorsal surface of thorax of <t>Prox1-GFP</t> control mouse after horseradish peroxidase (HRP) staining for GFP. Right panel: Drawing of whole mount where green lines show GFP + lymphatic vessels [thoracic duct (TD), paravertebral lymphatic vessel (PVL), and lymphatic plexus (LP)]. Gray lines mark the location of ribs. B: Paravertebral lymphatic vessel ( arrows indicate valves) and connecting lymphatic ( arrowheads indicate valves) to subpleural lymphatic plexuses. C: Fluorescence stereomicroscopic image of the pleural surface of the chest wall of a 3-week-old Prox1-GFP mouse showing lymphatic vessels (green; arrowheads ) that connect the TD to PVLs. Some regions of the connecting lymphatics ( arrow ) are obscured by overlying tissue. D: Images of a region of chest wall showing sites of tracer leakage (red; left panel ), LYVE-1–HRP staining of lymphatic plexuses (brown; middle panel ), and an overlay of the two (white; LYVE-1–HRP; right panel ). E: Enlarged images of lymphatic plexuses (LYVE-1–HRP) in chest wall of single transgenic control mouse and ADN–VEGF-C mouse, the latter showing bud-like outgrowths ( arrowheads ). F: Regional area per lymphatic plexus in single-transgenic and ADN–VEGF-C mice. Each dot shows the area of one plexus. Red lines indicate plexus area means. n = 7 mice ( D ); n = 4 mice per group ( E and F ). ∗∗ P
    Gfp, supplied by AvesLabs, used in various techniques. Bioz Stars score: 93/100, based on 1154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam fitc conjugated secondary antibodies
    Immunocytochemical assessment of surface phenotypes for KCs and MNCs under the influence of M1 or M2 inducers. Fluorescent microscopy images display immunostaining <t>(FITC,</t> green); cell nuclei are <t>counterstained</t> with DAPI (blue); KCs: Kupffer cells; MNCs: macrophages of monocytic lineage; scale bar, 50 μ m.
    Fitc Conjugated Secondary Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore monoclonal anti actin alpha smooth muscle fitc antibody
    Immunocytochemical assessment of surface phenotypes for KCs and MNCs under the influence of M1 or M2 inducers. Fluorescent microscopy images display immunostaining <t>(FITC,</t> green); cell nuclei are <t>counterstained</t> with DAPI (blue); KCs: Kupffer cells; MNCs: macrophages of monocytic lineage; scale bar, 50 μ m.
    Monoclonal Anti Actin Alpha Smooth Muscle Fitc Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 4 6 diamidino 2 phenylindole
    Effect of GLP on radiation-induced inhibition of HepG2 cell growth. HepG2 cells were treated with GLP (10 µM) for 72 h and then with radiation (6 Gy) for 30 min. Treated cells were stained with (A) DAPI (blue) and (B) anti-γ-H2AX antibody (red). GLP, Ganoderma lucidum polysaccharide; DAPI, 4′, <t>6-diamidino-2-phenylindole;</t> IR, irradiation; DMSO, dimethyl sulfoxide. *P
    4 6 Diamidino 2 Phenylindole, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 25556 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore triton x 100
    Effect of GLP on radiation-induced inhibition of HepG2 cell growth. HepG2 cells were treated with GLP (10 µM) for 72 h and then with radiation (6 Gy) for 30 min. Treated cells were stained with (A) DAPI (blue) and (B) anti-γ-H2AX antibody (red). GLP, Ganoderma lucidum polysaccharide; DAPI, 4′, <t>6-diamidino-2-phenylindole;</t> IR, irradiation; DMSO, dimethyl sulfoxide. *P
    Triton X 100, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 65173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore situ apoptosis detection kit
    Spermatocyte <t>apoptosis</t> in Brg1 KO testis. A ) Apoptotic cells were visualized using TUNEL assays detecting DNA breaks. B ) FACS analysis of apoptosis based on DNA content and cell morphology. Testis single cell suspension was stained with PI to reveal haploid
    Situ Apoptosis Detection Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam fluorescein isothiocyanate
    Association between gp78 and cidec in hepatic steatosis. (A) Nuclei were stained with DAPI, (a) gp78 was visualized (fluorescein <t>isothiocyanate</t> conjugated secondary antibody; red staining). (b) Cidec was visualized (tetraethyl rhodamine isothiocyanate conjugated secondary antibody; green staining); (c) Colocalization of gp78 and cidec (yellow) visualized on the surface of lipid droplets using immunofluorescent microscopy and identified by coimmunoprecipitation (scale bar=15 µm). (B) Gp78 and Cidec direct interaction detected by coimmunoprecipitation assay. gp78, glycoprotein; cidec, cell death-inducing DFFA-like effector c.
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    Abcam goat anti rabbit igg h l fitc
    Co-localization of IRF3 and HSPD1. A. HeLa cells were transfected with the MAVS or control plasmid. At 8 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 and mouse antibody against HSPD1 and further incubated with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L <t>(FITC).</t> Nuclei were stained with DAPI. B. HeLa cells were transfected with the MAVS or control plasmid. At 16 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 (phospho S386) and mouse antibody against HSPD1 and further developed with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L (FITC). Nuclei were stained with DAPI.
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    Millipore anti rabbit igg whole molecule fitc antibody
    Co-localization of IRF3 and HSPD1. A. HeLa cells were transfected with the MAVS or control plasmid. At 8 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 and mouse antibody against HSPD1 and further incubated with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L <t>(FITC).</t> Nuclei were stained with DAPI. B. HeLa cells were transfected with the MAVS or control plasmid. At 16 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 (phospho S386) and mouse antibody against HSPD1 and further developed with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L (FITC). Nuclei were stained with DAPI.
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    Abcam triton x 100
    Co-localization of IRF3 and HSPD1. A. HeLa cells were transfected with the MAVS or control plasmid. At 8 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 and mouse antibody against HSPD1 and further incubated with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L <t>(FITC).</t> Nuclei were stained with DAPI. B. HeLa cells were transfected with the MAVS or control plasmid. At 16 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 (phospho S386) and mouse antibody against HSPD1 and further developed with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L (FITC). Nuclei were stained with DAPI.
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    Image Search Results


    PNx induces increased nuclei numbers in α 1 +/− mice. Left ventricle sections from each experimental group were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and cardiac troponin I (cTnI, red) as described in materials

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Reduction of Na/K-ATPase affects cardiac remodeling and increases c-kit cell abundance in partial nephrectomized mice

    doi: 10.1152/ajpheart.00102.2014

    Figure Lengend Snippet: PNx induces increased nuclei numbers in α 1 +/− mice. Left ventricle sections from each experimental group were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and cardiac troponin I (cTnI, red) as described in materials

    Article Snippet: To count cardiac cell number, slides were stained for cardiac troponin I (cTnI, a cardiomyocyte specific marker, Cat. No. AB47003, Abcam) and 4′,6-diamidino-2-phenylindole (DAPI, a nuclear marker, Cat. No. D1306, Life Technologies).

    Techniques: Mouse Assay, Staining

    Nuclear translocation activity of wild-type Angiogenin-GST and mutants. HeLa cells, untreated or incubated with 2 µg/ml of wild-type Angiogenin-GST, or the D22G, L35P mutants were fixed, permeabilized and stained with mouse anti-Angiogenin monoclonal antibody and Alexa Fluor 555 goat anti-mouse IgG, while the nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride. Individual channels, as well as a merge, are shown. The magnification in all cases is 200X. The images are representative of cells from at least three areas (each area containing 35–50 cells) from two independent experiments.

    Journal: PLoS ONE

    Article Title: Computational and Functional Characterization of Angiogenin Mutations, and Correlation with Amyotrophic Lateral Sclerosis

    doi: 10.1371/journal.pone.0111963

    Figure Lengend Snippet: Nuclear translocation activity of wild-type Angiogenin-GST and mutants. HeLa cells, untreated or incubated with 2 µg/ml of wild-type Angiogenin-GST, or the D22G, L35P mutants were fixed, permeabilized and stained with mouse anti-Angiogenin monoclonal antibody and Alexa Fluor 555 goat anti-mouse IgG, while the nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride. Individual channels, as well as a merge, are shown. The magnification in all cases is 200X. The images are representative of cells from at least three areas (each area containing 35–50 cells) from two independent experiments.

    Article Snippet: The nuclei were counter-stained with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride (Molecular Probes).

    Techniques: Translocation Assay, Activity Assay, Incubation, Staining

    IKKβ and IKKε phosphorylate YAP. ( a ) 293T cells were co-transfected with GFP-YAP and Flag-IKKα, HA-IKKβ or IKKε. The cell lysates were analyzed by western blot with indicated antibodies. The asterisk showed the shifted band ( b , c ) 293T cells were co-transfected with GFP-YAP and HA-IKKβ or IKKε. Cell lysates were immunoprecipitated with indicated antibodies and analyzed by western blot. ( d ) 293T cells were transfected with GFP-YAP and HA-IKKβ or IKKε. The cell lysates were analyzed by phos-tag gel western blot. The asterisk showed the phosphorylated band. ( e ) The recombinant GST-YAP protein was incubated with immunoprecipitated HA-IKKβ or IKKε in phosphorylation buffer. Reactions were subjected to electrophoresis and immunoblotted with antibody against pan-phosphorylated serine/threonine (S/T). ( f ) MCF7 cells were treated with TNFα for indicated times, the cells were then lysed for phos-tag gel western blot. The asterisk showed the phosphorylated band. ( g ) 293T cells were transfected with GFP-YAP and HA-IKKβ/ε. Cell lysates were immunoprecipitated with GFP antibody followed by immunoblotting with TEAD4 antibody. ( h ) MCF7 cells were treated with 10 μ M TPCA-1 or 10 μ M IKK-16 for 12 h. Then endogenous YAP was immunoprecipitated and followed by immunoblotting with TEAD4 antibody.

    Journal: Oncogenesis

    Article Title: TNFα-YAP/p65-HK2 axis mediates breast cancer cell migration

    doi: 10.1038/oncsis.2017.83

    Figure Lengend Snippet: IKKβ and IKKε phosphorylate YAP. ( a ) 293T cells were co-transfected with GFP-YAP and Flag-IKKα, HA-IKKβ or IKKε. The cell lysates were analyzed by western blot with indicated antibodies. The asterisk showed the shifted band ( b , c ) 293T cells were co-transfected with GFP-YAP and HA-IKKβ or IKKε. Cell lysates were immunoprecipitated with indicated antibodies and analyzed by western blot. ( d ) 293T cells were transfected with GFP-YAP and HA-IKKβ or IKKε. The cell lysates were analyzed by phos-tag gel western blot. The asterisk showed the phosphorylated band. ( e ) The recombinant GST-YAP protein was incubated with immunoprecipitated HA-IKKβ or IKKε in phosphorylation buffer. Reactions were subjected to electrophoresis and immunoblotted with antibody against pan-phosphorylated serine/threonine (S/T). ( f ) MCF7 cells were treated with TNFα for indicated times, the cells were then lysed for phos-tag gel western blot. The asterisk showed the phosphorylated band. ( g ) 293T cells were transfected with GFP-YAP and HA-IKKβ/ε. Cell lysates were immunoprecipitated with GFP antibody followed by immunoblotting with TEAD4 antibody. ( h ) MCF7 cells were treated with 10 μ M TPCA-1 or 10 μ M IKK-16 for 12 h. Then endogenous YAP was immunoprecipitated and followed by immunoblotting with TEAD4 antibody.

    Article Snippet: Antibodies against β-actin, GFP, Myc, Flag, HA and TEAD4 were purchased from Sigma-Aldrich.

    Techniques: Transfection, Western Blot, Immunoprecipitation, Recombinant, Incubation, Electrophoresis

    YAP-TEAD and p65 synergistically regulate the expression of HK2. ( a ) MCF7 cell lines stably expressing shRNA (1# and 2#) targeting p65 were treated with control or 10 μ M TNFα or macrophage CM for 24 h, p65 knockdown efficiency and HK2 protein level were confirmed by western blot. ( b ) 293T cells were co-transfected with GFP-YAP and Myc-p65. Cell lysates were immunoprecipitated with Myc antibody and then analyzed by western blot. ( c ) 293T cells were co-transfected with GFP-p65 and Flag-YAP. Cell lysates were immunoprecipitated with Flag antibody and then analyzed by western blot. ( d ) 293T cells were transfected with Myc-p65 and HA-TEAD4. Immunoprecipitation of Myc-p65 and co-immunoprecipitation of HA-TEAD4 were detected by western blot. ( e ) MCF7 cells were treated with 10 μ M TNFα for 12 h, cell lysates were immunopreipitated with rabbit IgG or YAP antibody, the endogous co- immunopreipitated p65 was measured by immunoblotting. ( f ) 293T cells were co-transfected with GFP-YAP and Myc-p65. After transfection, the cells were treated with 10 μ M TPCA-1 or 10 μ M IKK-16 for followed 18 h, Myc-p65 was immunoprecipitated with Myc antibody and the co-immunoprecipitated GFP-YAP was determined by western blot. ( g ) MCF7 cells were treated with 10 μ M TNFα for 12 h. The cell lysates were used for ChIP analysis with antibody against TEAD4, YAP or p65. The binding of TEAD4, YAP or p65 on HK2 promoter were detected by real-time PCR. ( h , i ) Control and shYAP MCF7 cells were treated with TNFα for 12 h. The mRNA levels of CCL2 ( h ) and IL-6 ( i ) were analyzed by real-time PCR. ( j , k ) MCF7 cells were treated with 10 μ M TNFα for 12 h. Then, the cell lysates were used for ChIP analysis with antibody against TEAD4, YAP or p65. The binding of TEAD4, YAP or p65 on CCL2 or IL-6 promoter were detected by real-time PCR. ( l ) The model of YAP/TEAD/p65 interact with each other to synergistically regulate gene transcription and breast cancer cell migration under TNFα treatment. The error bars represent the means±s.d. (* P

    Journal: Oncogenesis

    Article Title: TNFα-YAP/p65-HK2 axis mediates breast cancer cell migration

    doi: 10.1038/oncsis.2017.83

    Figure Lengend Snippet: YAP-TEAD and p65 synergistically regulate the expression of HK2. ( a ) MCF7 cell lines stably expressing shRNA (1# and 2#) targeting p65 were treated with control or 10 μ M TNFα or macrophage CM for 24 h, p65 knockdown efficiency and HK2 protein level were confirmed by western blot. ( b ) 293T cells were co-transfected with GFP-YAP and Myc-p65. Cell lysates were immunoprecipitated with Myc antibody and then analyzed by western blot. ( c ) 293T cells were co-transfected with GFP-p65 and Flag-YAP. Cell lysates were immunoprecipitated with Flag antibody and then analyzed by western blot. ( d ) 293T cells were transfected with Myc-p65 and HA-TEAD4. Immunoprecipitation of Myc-p65 and co-immunoprecipitation of HA-TEAD4 were detected by western blot. ( e ) MCF7 cells were treated with 10 μ M TNFα for 12 h, cell lysates were immunopreipitated with rabbit IgG or YAP antibody, the endogous co- immunopreipitated p65 was measured by immunoblotting. ( f ) 293T cells were co-transfected with GFP-YAP and Myc-p65. After transfection, the cells were treated with 10 μ M TPCA-1 or 10 μ M IKK-16 for followed 18 h, Myc-p65 was immunoprecipitated with Myc antibody and the co-immunoprecipitated GFP-YAP was determined by western blot. ( g ) MCF7 cells were treated with 10 μ M TNFα for 12 h. The cell lysates were used for ChIP analysis with antibody against TEAD4, YAP or p65. The binding of TEAD4, YAP or p65 on HK2 promoter were detected by real-time PCR. ( h , i ) Control and shYAP MCF7 cells were treated with TNFα for 12 h. The mRNA levels of CCL2 ( h ) and IL-6 ( i ) were analyzed by real-time PCR. ( j , k ) MCF7 cells were treated with 10 μ M TNFα for 12 h. Then, the cell lysates were used for ChIP analysis with antibody against TEAD4, YAP or p65. The binding of TEAD4, YAP or p65 on CCL2 or IL-6 promoter were detected by real-time PCR. ( l ) The model of YAP/TEAD/p65 interact with each other to synergistically regulate gene transcription and breast cancer cell migration under TNFα treatment. The error bars represent the means±s.d. (* P

    Article Snippet: Antibodies against β-actin, GFP, Myc, Flag, HA and TEAD4 were purchased from Sigma-Aldrich.

    Techniques: Expressing, Stable Transfection, shRNA, Western Blot, Transfection, Immunoprecipitation, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Migration

    Specialization of Sema3a + cardiomyocytes into the conduction system in the developing heart. ( a – f ) Z-stack images of RFP and GFP immunostaining on Sema3a-CreERT2; R26-tdTomato; Cx40-GFP heart sections. Tamoxifen was administered at E12.5 ( a , b ), E14.5 ( c , d ) and E18.5 ( e , f ). The hearts were collected at P7 and P21 for each group. YZ indicates signals from the dotted lines on the Z-stack images. Scale bars, 50 µm. Each image is representative of 5 individual samples. ( g ) Schematic figure showing Sema3a + cells (red) and Cx40 + cells (green) in the developing and adult heart.

    Journal: Scientific Reports

    Article Title: Genetic targeting of Purkinje fibres by Sema3a-CreERT2

    doi: 10.1038/s41598-018-20829-9

    Figure Lengend Snippet: Specialization of Sema3a + cardiomyocytes into the conduction system in the developing heart. ( a – f ) Z-stack images of RFP and GFP immunostaining on Sema3a-CreERT2; R26-tdTomato; Cx40-GFP heart sections. Tamoxifen was administered at E12.5 ( a , b ), E14.5 ( c , d ) and E18.5 ( e , f ). The hearts were collected at P7 and P21 for each group. YZ indicates signals from the dotted lines on the Z-stack images. Scale bars, 50 µm. Each image is representative of 5 individual samples. ( g ) Schematic figure showing Sema3a + cells (red) and Cx40 + cells (green) in the developing and adult heart.

    Article Snippet: The primary antibodies and dilution ratios were as follows: RFP (Rockland, 600-401-379,1:500); ESR (Abcam, ab27595, prediluted); GFP (Nacalai Tesque, 04404–84, 1:100); GFP (Abcam, ab6662, 1:200); TNNI3 (Abcam, ab56357, 1:100); Cx40 (Alpha Diagnostic, Cx40-A, 1:100); HCN4 (Abcam, ab32675, 1:100); tyrosine hydroxylase (TH, Millipore, AB152, 1:100); and Tuj1 (Covance, MMS-435P, 1:100).

    Techniques: Immunostaining

    The adult expression map of Sema3a in the heart. ( a ) Schematic showing the crossing of the mice to generate the Sema3a-CreERT2; R26-tdTomato mice. ( b ) Genetic labelling of the Sema3a + cells via tamoxifen administration. ( c ) Whole-mount fluorescence and bright-field views of a Sema3a-CreERT2; R26-tdTomato mouse heart. ( d ) Immunostaining for RFP and TNNI3 on a Sema3a-CreERT2; R26-tdTomato heart section showing the rarity of RFP + cells in the atrium. LA, left atrium. ( e ) No Sema3a + cells were detected in the SA node. ( f ) The expression of Sema3a in the AV node. ( g ) Immunostaining for RFP, GFP and TNNI3 in a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart section showing that the CX40 + coronary artery (arrowhead) was negative for RFP. ( h ) Whole-mount fluorescence view of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart. LBB, left bundle branch; LPF, left Purkinje fibre; IVS, interventricular septum; LVW, left ventricular free wall. The dotted line indicates the limits between the IVS and the LVW. ( i ) Immunostaining for RFP and GFP on heart sections of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse. Sema3a was not detected in the LBB or RBB, which were positive for Cx40-GFP. ( j ) Z-stack confocal image showing that Sema3a was expressed in the Purkinje fibres. XZ and YZ indicate the signals from the dotted lines on the Z-stack images in ( j ). Scale bars, 1 mm in ( c ) 500 µm in ( e , g , h ) and 100 µm in ( d , f , i ) and ( j ). Each image is representative of 5 individual samples.

    Journal: Scientific Reports

    Article Title: Genetic targeting of Purkinje fibres by Sema3a-CreERT2

    doi: 10.1038/s41598-018-20829-9

    Figure Lengend Snippet: The adult expression map of Sema3a in the heart. ( a ) Schematic showing the crossing of the mice to generate the Sema3a-CreERT2; R26-tdTomato mice. ( b ) Genetic labelling of the Sema3a + cells via tamoxifen administration. ( c ) Whole-mount fluorescence and bright-field views of a Sema3a-CreERT2; R26-tdTomato mouse heart. ( d ) Immunostaining for RFP and TNNI3 on a Sema3a-CreERT2; R26-tdTomato heart section showing the rarity of RFP + cells in the atrium. LA, left atrium. ( e ) No Sema3a + cells were detected in the SA node. ( f ) The expression of Sema3a in the AV node. ( g ) Immunostaining for RFP, GFP and TNNI3 in a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart section showing that the CX40 + coronary artery (arrowhead) was negative for RFP. ( h ) Whole-mount fluorescence view of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse heart. LBB, left bundle branch; LPF, left Purkinje fibre; IVS, interventricular septum; LVW, left ventricular free wall. The dotted line indicates the limits between the IVS and the LVW. ( i ) Immunostaining for RFP and GFP on heart sections of a Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse. Sema3a was not detected in the LBB or RBB, which were positive for Cx40-GFP. ( j ) Z-stack confocal image showing that Sema3a was expressed in the Purkinje fibres. XZ and YZ indicate the signals from the dotted lines on the Z-stack images in ( j ). Scale bars, 1 mm in ( c ) 500 µm in ( e , g , h ) and 100 µm in ( d , f , i ) and ( j ). Each image is representative of 5 individual samples.

    Article Snippet: The primary antibodies and dilution ratios were as follows: RFP (Rockland, 600-401-379,1:500); ESR (Abcam, ab27595, prediluted); GFP (Nacalai Tesque, 04404–84, 1:100); GFP (Abcam, ab6662, 1:200); TNNI3 (Abcam, ab56357, 1:100); Cx40 (Alpha Diagnostic, Cx40-A, 1:100); HCN4 (Abcam, ab32675, 1:100); tyrosine hydroxylase (TH, Millipore, AB152, 1:100); and Tuj1 (Covance, MMS-435P, 1:100).

    Techniques: Expressing, Mouse Assay, Fluorescence, Immunostaining

    Dox-dependent TS expression in TFTS66 cells. A.  TS antigens in cell lysates of TFTS66 transformant were detected by immunoblotting using anti-human TS mouse monoclonal antibody. The standard cell lysates (0 ng/ml Dox) were titrated, and a standard curve for detection was obtained from the signal intensity on the digitized image (upper panel). Using the highly linear detection characteristics (p = 0.997), TS expression levels were quantified: rectangle, TFTS66; circle, the control transformant, TFC7.  B.  TS expression in TFTS66 cells exposed to various concentrations of Dox was assessed by immunoblotting and similarly quantified. The symbols are shaded according to the Dox concentrations. Some of the data points are also shown in Fig 2A.  C.  The quantity of TS protein (TS total , see   Materials and methods ) and its catalytic activity were enzymatically assayed in lysates prepared from TFTS66 cells exposed to 0, 0.5 and 1.0 ng/ml Dox. The results are plotted as a function of the TS expression level determined by immunoblotting: open rectangle, Dox0; shaded rectangle, Dox0.5; closed rectangle, Dox1.0.  D.  TS expression in TFTS66 cells was observed using fluorescent immunocytochemistry. Cells grown on chamber slides were fixed and reacted with TS-specific antibody. Cellular distribution of TS antigens was visualized by red fluorescent signals. Cells were also counterstained with Hoechst 33342. Results obtained in TFTS66 (0, 0.1 and 1.0 ng/ml Dox) and its parental line, DLD-1, are shown (magnification X100).

    Journal: PLoS ONE

    Article Title: Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

    doi: 10.1371/journal.pone.0123076

    Figure Lengend Snippet: Dox-dependent TS expression in TFTS66 cells. A. TS antigens in cell lysates of TFTS66 transformant were detected by immunoblotting using anti-human TS mouse monoclonal antibody. The standard cell lysates (0 ng/ml Dox) were titrated, and a standard curve for detection was obtained from the signal intensity on the digitized image (upper panel). Using the highly linear detection characteristics (p = 0.997), TS expression levels were quantified: rectangle, TFTS66; circle, the control transformant, TFC7. B. TS expression in TFTS66 cells exposed to various concentrations of Dox was assessed by immunoblotting and similarly quantified. The symbols are shaded according to the Dox concentrations. Some of the data points are also shown in Fig 2A. C. The quantity of TS protein (TS total , see Materials and methods ) and its catalytic activity were enzymatically assayed in lysates prepared from TFTS66 cells exposed to 0, 0.5 and 1.0 ng/ml Dox. The results are plotted as a function of the TS expression level determined by immunoblotting: open rectangle, Dox0; shaded rectangle, Dox0.5; closed rectangle, Dox1.0. D. TS expression in TFTS66 cells was observed using fluorescent immunocytochemistry. Cells grown on chamber slides were fixed and reacted with TS-specific antibody. Cellular distribution of TS antigens was visualized by red fluorescent signals. Cells were also counterstained with Hoechst 33342. Results obtained in TFTS66 (0, 0.1 and 1.0 ng/ml Dox) and its parental line, DLD-1, are shown (magnification X100).

    Article Snippet: Fixed cells were next reacted with 2 μg/ml anti-human TS mouse monoclonal antibody TS106 (Abcam plc, Cambridge, United Kingdom) at RT for 1h, followed by washing with PBS supplemented with 0.5% BSA and 0.15% glycine (PBG), and then incubated with 1:100 diluted secondary antibody, Qdot 655 goat F(ab’)2 anti-mouse IgG conjugate (H+L) (Life Technologies), at RT for 1 h. After washing, cells were stained with 0.01% Hoechst 33342 (Life Technologies) and mounted using Fluorescent Mounting Medium (Dako Denmark A/S, Glostrup, Denmark).

    Techniques: Expressing, Activity Assay, Immunocytochemistry

    AnkB and AnkG interact with NF155 in vivo and can be targeted to paranodes independently of paranodal junctions and NF155. ( a–d ) P8 sciatic nerve sections were stained for AnkB (N105/17), NFasc and Caspr ( a, b ), and the respective longitudinal line scans are shown ( c, d ). ( e ) Immunoprecipitation of AnkB from adult rat sciatic nerves co-precipitated NF155. Immunoprecipitation with the anti-GFP antibody served as a negative control. Heavy chain, mouse IgG heavy chain. AnkB was detected by H-300 rabbit polyclonal antibodies. ( f ) Immunoprecipitation of AnkG from P21 mouse spinal cords co-precipitated NF186 and NF155. AnkG was detected by the goat polyclonal antibodies. The immunoprecipitation in ( e, f ) was reproduced at least three times. The full blots are presented in Supplementary Fig. 3 . ( g–i ) Immunostaining of P7 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the residual AnkB and NFasc at paranodes. ( j–l ) Immunostaining of P7 spinal cords (AnkG, N106/36). Arrows point to the residual AnkG and NFasc at paranodes. ( m–o ) Immunostaining of P5 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the paranodes with residual AnkB. ( p–r ) Immunostaining of P12 Nfasc-cHet ( Cnp-Cre;Nfasc f/+ ) and Nfasc-cKO ( Cnp-Cre;Nfasc f/f ) spinal cords (AnkG, N106/36). Two mice per genotype and more than 100 nodes were examined in each mouse. 80–190 nodes per animal were quantified. Scale bars = ( a, b ) 5 μm for (a) and 3.3 μm for (b); 5 μm ( g–i , j–l , m–o , and p–r ).

    Journal: Nature neuroscience

    Article Title: GLIAL ANKYRINS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY

    doi: 10.1038/nn.3858

    Figure Lengend Snippet: AnkB and AnkG interact with NF155 in vivo and can be targeted to paranodes independently of paranodal junctions and NF155. ( a–d ) P8 sciatic nerve sections were stained for AnkB (N105/17), NFasc and Caspr ( a, b ), and the respective longitudinal line scans are shown ( c, d ). ( e ) Immunoprecipitation of AnkB from adult rat sciatic nerves co-precipitated NF155. Immunoprecipitation with the anti-GFP antibody served as a negative control. Heavy chain, mouse IgG heavy chain. AnkB was detected by H-300 rabbit polyclonal antibodies. ( f ) Immunoprecipitation of AnkG from P21 mouse spinal cords co-precipitated NF186 and NF155. AnkG was detected by the goat polyclonal antibodies. The immunoprecipitation in ( e, f ) was reproduced at least three times. The full blots are presented in Supplementary Fig. 3 . ( g–i ) Immunostaining of P7 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the residual AnkB and NFasc at paranodes. ( j–l ) Immunostaining of P7 spinal cords (AnkG, N106/36). Arrows point to the residual AnkG and NFasc at paranodes. ( m–o ) Immunostaining of P5 sciatic nerves (AnkB, rabbit polyclonal). Arrows point to the paranodes with residual AnkB. ( p–r ) Immunostaining of P12 Nfasc-cHet ( Cnp-Cre;Nfasc f/+ ) and Nfasc-cKO ( Cnp-Cre;Nfasc f/f ) spinal cords (AnkG, N106/36). Two mice per genotype and more than 100 nodes were examined in each mouse. 80–190 nodes per animal were quantified. Scale bars = ( a, b ) 5 μm for (a) and 3.3 μm for (b); 5 μm ( g–i , j–l , m–o , and p–r ).

    Article Snippet: Antibodies The primary antibodies used were: rabbit antibodies against AnkB membrane-binding domain , AnkB C-terminal region (H-300, SantaCruz), AnkG spectrin-binding domain and C-terminal region , AnkG exon 1b and exon 1e (generated by immunizing against peptides unique to these domains followed by affinity purification of specific antibodies), AnkG 480/270 (generated, affinity purified, and kindly provided by Dr. Paul Jenkins, Duke University), AnkR (generated against His-tagged, full-length human AnkR and affinity purified), GFP (A11122, Life Technologies), NF-M (AB1987, EMD Millipore), Caspr , βIV spectrin SD , Kv1.2 , Caspr2 (ab33994, Abcam), Nav1.2 , Nav1.6 , and 4.1G (kindly provided by Dr. Elior Peles); mouse monoclonal antibodies against AnkB membrane-binding domain (N105/13 and N105/17, UC Davis/NIH NeuroMab Facility), AnkB spectrin-binding domain (2.20, SantaCruz), AnkG spectrin-binding domain or C-terminal region (N106/36 and N106/65, NeuroMab), Caspr (K65/35, NeuroMab), contactin (K73/20, NeuroMab), actin (C4, EMD Millipore), ZO-1 (ZO1-1A12, Life Technologies), E-cadherin (4A2C7, Life Technologies), Kv1.2 (K14/16, NeuroMab), Nav1.6 (K87A/10, NeuroMab), CNP (11-5B, Sigma-Aldrich), MAG (513, EMD Millipore), MBP (SMI-94, Covance), Necl4 (N244/5, NeuroMab), pan-Nav channels (K58/35 ) and GFP (N86/38, NeuroMab); the rat monoclonal antibody against MBP (MAB386, EMD Millipore); chicken antibodies against NFasc (AF3235, R & D Systems) and βIV spectrin SD ; goat antibodies against contactin (AF904, R & D Systems), connexin 32 (C-20, Santa Cruz), and AnkG (generated against the C-terminal domain of AnkG minus the death domain and affinity purified).

    Techniques: In Vivo, Staining, Immunoprecipitation, Negative Control, Immunostaining, Mouse Assay

    Paranodal AnkB is derived from Schwann cells in the PNS. ( a ) Immunostaining of a mouse sciatic nerve for AnkG (node, rabbit polyclonal anti-AnkG) and AnkB (paranodes, N105/17). ( b ) Cultured DRG neurons were infected with adenovirus carrying a GFP and AnkB shRNA-expressing construct, and immunostained (AnkB, N105/13). Arrowheads point to the GFP-positive axon. ( c, d ) Schwann cells were added to the same culture as in ( b ) and induced to myelinate. The co-culture was labeled for myelin basic protein (MBP), GFP and AnkB (N105/13 ( c ) or N105/17 ( d )). The arrows point to paranodal AnkB. A line scan of fluorescence intensity of the paranode indicated in ( d ) is shown in the inset. ( e ) Immunoblots of lysates from rat hippocampal (Hc) neuron and purified Schwann cell (Sc) cultures (AnkB, N105/17). The full blots are presented in Supplementary Fig. 3 . ( f ) DRG neurons from the AnkB conventional KO were co-cultured with myelinating rat Schwann cells and immunostained for AnkB (N105/17), neurofilament-M (NF-M) and MBP. The arrow points to a paranode. Localization of AnkB along the inner mesaxon 13 was also observed as spiral extensions from paranodal junctions. ( g ) The scheme of the Ank2 conditional allele. The two loxP sites (red triangles) flank exon 24. After Cre recombination and removal of exon 24, a premature stop codon is generated in exon 25. ( h, i ) Immunostaining of 4-week-old AnkB-cHet ( h ) and AnkB-cKO ( i ) sciatic nerves (AnkB, rabbit polyclonal anti-AnkB). Arrows point to paranodal junctions. Scale bars = 5 μm ( a; h, i ), and 10 μm ( b–d, f ).

    Journal: Nature neuroscience

    Article Title: GLIAL ANKYRINS FACILITATE PARANODAL AXOGLIAL JUNCTION ASSEMBLY

    doi: 10.1038/nn.3858

    Figure Lengend Snippet: Paranodal AnkB is derived from Schwann cells in the PNS. ( a ) Immunostaining of a mouse sciatic nerve for AnkG (node, rabbit polyclonal anti-AnkG) and AnkB (paranodes, N105/17). ( b ) Cultured DRG neurons were infected with adenovirus carrying a GFP and AnkB shRNA-expressing construct, and immunostained (AnkB, N105/13). Arrowheads point to the GFP-positive axon. ( c, d ) Schwann cells were added to the same culture as in ( b ) and induced to myelinate. The co-culture was labeled for myelin basic protein (MBP), GFP and AnkB (N105/13 ( c ) or N105/17 ( d )). The arrows point to paranodal AnkB. A line scan of fluorescence intensity of the paranode indicated in ( d ) is shown in the inset. ( e ) Immunoblots of lysates from rat hippocampal (Hc) neuron and purified Schwann cell (Sc) cultures (AnkB, N105/17). The full blots are presented in Supplementary Fig. 3 . ( f ) DRG neurons from the AnkB conventional KO were co-cultured with myelinating rat Schwann cells and immunostained for AnkB (N105/17), neurofilament-M (NF-M) and MBP. The arrow points to a paranode. Localization of AnkB along the inner mesaxon 13 was also observed as spiral extensions from paranodal junctions. ( g ) The scheme of the Ank2 conditional allele. The two loxP sites (red triangles) flank exon 24. After Cre recombination and removal of exon 24, a premature stop codon is generated in exon 25. ( h, i ) Immunostaining of 4-week-old AnkB-cHet ( h ) and AnkB-cKO ( i ) sciatic nerves (AnkB, rabbit polyclonal anti-AnkB). Arrows point to paranodal junctions. Scale bars = 5 μm ( a; h, i ), and 10 μm ( b–d, f ).

    Article Snippet: Antibodies The primary antibodies used were: rabbit antibodies against AnkB membrane-binding domain , AnkB C-terminal region (H-300, SantaCruz), AnkG spectrin-binding domain and C-terminal region , AnkG exon 1b and exon 1e (generated by immunizing against peptides unique to these domains followed by affinity purification of specific antibodies), AnkG 480/270 (generated, affinity purified, and kindly provided by Dr. Paul Jenkins, Duke University), AnkR (generated against His-tagged, full-length human AnkR and affinity purified), GFP (A11122, Life Technologies), NF-M (AB1987, EMD Millipore), Caspr , βIV spectrin SD , Kv1.2 , Caspr2 (ab33994, Abcam), Nav1.2 , Nav1.6 , and 4.1G (kindly provided by Dr. Elior Peles); mouse monoclonal antibodies against AnkB membrane-binding domain (N105/13 and N105/17, UC Davis/NIH NeuroMab Facility), AnkB spectrin-binding domain (2.20, SantaCruz), AnkG spectrin-binding domain or C-terminal region (N106/36 and N106/65, NeuroMab), Caspr (K65/35, NeuroMab), contactin (K73/20, NeuroMab), actin (C4, EMD Millipore), ZO-1 (ZO1-1A12, Life Technologies), E-cadherin (4A2C7, Life Technologies), Kv1.2 (K14/16, NeuroMab), Nav1.6 (K87A/10, NeuroMab), CNP (11-5B, Sigma-Aldrich), MAG (513, EMD Millipore), MBP (SMI-94, Covance), Necl4 (N244/5, NeuroMab), pan-Nav channels (K58/35 ) and GFP (N86/38, NeuroMab); the rat monoclonal antibody against MBP (MAB386, EMD Millipore); chicken antibodies against NFasc (AF3235, R & D Systems) and βIV spectrin SD ; goat antibodies against contactin (AF904, R & D Systems), connexin 32 (C-20, Santa Cruz), and AnkG (generated against the C-terminal domain of AnkG minus the death domain and affinity purified).

    Techniques: Derivative Assay, Immunostaining, Cell Culture, Infection, shRNA, Expressing, Construct, Co-Culture Assay, Labeling, Fluorescence, Western Blot, Purification, Generated

    Molecular characterization of the pocket. pH Smo cilia ( green ) were fixed and first labeled with anti-GFP ( red ) in unpermeabilized cells to label the extracellular region ( arrowhead ) and then fixed again, permeabilized and stained with various ciliary markers ( blue ). a Acetylated tubulin (Ac Tub) stains the entire length of the ciliary axoneme and does not correlate with anti-GFP staining. b The anti-GFP staining ends above the basal body (BB), marked by pericentrin. c The anti-GFP staining also ends above the transition zone (TZ), marked by CEP290. d – f The anti-GFP staining inversely coincides with: EHD1 ( d ), Septin 9 ( e ) and glutamylated tubulin (Glu Tub) ( f )

    Journal: Cilia

    Article Title: The IN/OUT assay: a new tool to study ciliogenesis

    doi: 10.1186/s13630-016-0044-2

    Figure Lengend Snippet: Molecular characterization of the pocket. pH Smo cilia ( green ) were fixed and first labeled with anti-GFP ( red ) in unpermeabilized cells to label the extracellular region ( arrowhead ) and then fixed again, permeabilized and stained with various ciliary markers ( blue ). a Acetylated tubulin (Ac Tub) stains the entire length of the ciliary axoneme and does not correlate with anti-GFP staining. b The anti-GFP staining ends above the basal body (BB), marked by pericentrin. c The anti-GFP staining also ends above the transition zone (TZ), marked by CEP290. d – f The anti-GFP staining inversely coincides with: EHD1 ( d ), Septin 9 ( e ) and glutamylated tubulin (Glu Tub) ( f )

    Article Snippet: The following antibodies were used: GFP (rabbit polyclonal; Invitrogen), GFP (mouse monoclonal; Invitrogen), acetylated α-tubulin (mouse monoclonal; Sigma-Aldrich), Arl13b (mouse monoclonal; NeuroMab), CEP290 (rabbit monoclonal; Bethyl), pericentrin (rabbit polyclonal; Covance), EHD1 (rabbit monoclonal; Abcam), glutamylated tubulin (rabbit polyclonal; Chemicon Int.), Septin 9 (rabbit polyclonal, Sigma-Aldrich), AlexaFluor 568 goat anti-rabbit (Invitrogen) and Atto 647 N goat anti-mouse (Sigma-Aldrich).

    Techniques: Labeling, Staining

    BC transplantation rescued mouse pulmonary function . (A) Injured mouse lung without or with GFP-labeled SOX9 + BCs transplantation by anti-GFP and anti-Fibronectin co-staining. Scale bar, 200 μm. (B) Left, immunofluorescence image of injured mouse lung transplanted with GFP-labeled SOX9 + BCs; right, immunostaining on the same section showing exclusion of α-SMA+ myofibroblasts from GFP + area. Scale bar, 200 μm. (C) CO 2 partial pressure of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse. (D) O 2 partial pressure of mouse arterial blood 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse. (E) O 2 saturation of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse

    Journal: Protein & Cell

    Article Title: Regeneration of functional alveoli by adult human SOX9+ airway basal cell transplantation

    doi: 10.1007/s13238-018-0506-y

    Figure Lengend Snippet: BC transplantation rescued mouse pulmonary function . (A) Injured mouse lung without or with GFP-labeled SOX9 + BCs transplantation by anti-GFP and anti-Fibronectin co-staining. Scale bar, 200 μm. (B) Left, immunofluorescence image of injured mouse lung transplanted with GFP-labeled SOX9 + BCs; right, immunostaining on the same section showing exclusion of α-SMA+ myofibroblasts from GFP + area. Scale bar, 200 μm. (C) CO 2 partial pressure of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse. (D) O 2 partial pressure of mouse arterial blood 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse. (E) O 2 saturation of mouse arterial blood before and 1 month after bleomycin-induced injury with or without SOX9 + BCs transplantation. Each dot indicates an individual mouse

    Article Snippet: Primary antibodies used in this work include BC markers:KRT5 (1:200, EP1601Y, Thermo), P63 (deltaN, 1:200, 4A4, Abcam), E-cadherin (1:200, H-108, Santa cruz), SOX9 (1:200, ERP14335-78, Abcam), SOX9 (1:200, AF3075, R & D); AEC markers: AQP5 (1:1000, EPR3747, Abcam), HOPX (1:500, E-1, Santa Cruz), PDPN (1:500, FL-162, Santa Cruz), SPC (1:200, M-20, Santa Cruz), SPC (1:200, FL-197, Santa Cruz), LAMP3 (1:200, 12632-1-AP, Proteintech); bronchiolar cell markers: CC10 (1:200, T-18, Santa Cruz), acetylated-α-Tubulin (1:1000, 6-11B-1, Abcam), MUC5AC (1:500, 45M1, Thermo), FOXJ1 (1:200, 2A5, eBioscience); vasculature markers: CD31 (1:100, M-20, Santa Cruz), CD34 (1:1000, EP373Y, Abcam); myofibroblast marker: α-SMA (1:500, 1A4, DAKO), Fibronectin (1:500, F14, Abcam), others: KI67 (1:200, RM-9106, Thermo), GFP (1:200, B-2, Santa Cruz), GFP (1:200, FL, Santa Cruz), GFP (1:200, T-19, Santa Cruz), ITGB1 (1:500, ERP16895, Abcam), Human specific Lamin A+C (1:200, EPR4100, Abcam).

    Techniques: Transplantation Assay, Labeling, Staining, Immunofluorescence, Immunostaining

    Regenerated alveoli with functional epithelium-capillary system . (A) Transplanted SOX9 + BCs (anti-GFP) and capillary endothelium marker (anti-CD34). Scale bar, 100 μm. (B) Confocal image of SOX9 + BCs regenerated alveoli (Alv) and the neighboring capillary blood vessel (Bv). Left, immunofluorescence; right, bright field. Scale bar, 20 μm. (C) Confocal image showing the basement membrane (ITGB1 + , white color, arrowhead indicated) between regenerated alveoli epithelium and capillary endothelium (CD31 + ). Scale bar, 10 μm. (D) Confocal image showing the cell adherens junction (E-cadherin + , white color) between regenerated alveoli epithelial cells. Scale bar, 20 μm. (E) Confocal image showing the cell tight junction (ZO-1 + ) between regenerated alveoli epithelial cells. Scale bar, 20 μm. (F) Direct fluorescence image of the transplanted GFP-labeled SOX9 + BCs (green) and bright-field image of tail vein delivered gold nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm. (G) Direct fluorescence image of the transplanted GFP-labeled SOX9 + BCs (green) and bright-field image of intratracheally delivered gold nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm

    Journal: Protein & Cell

    Article Title: Regeneration of functional alveoli by adult human SOX9+ airway basal cell transplantation

    doi: 10.1007/s13238-018-0506-y

    Figure Lengend Snippet: Regenerated alveoli with functional epithelium-capillary system . (A) Transplanted SOX9 + BCs (anti-GFP) and capillary endothelium marker (anti-CD34). Scale bar, 100 μm. (B) Confocal image of SOX9 + BCs regenerated alveoli (Alv) and the neighboring capillary blood vessel (Bv). Left, immunofluorescence; right, bright field. Scale bar, 20 μm. (C) Confocal image showing the basement membrane (ITGB1 + , white color, arrowhead indicated) between regenerated alveoli epithelium and capillary endothelium (CD31 + ). Scale bar, 10 μm. (D) Confocal image showing the cell adherens junction (E-cadherin + , white color) between regenerated alveoli epithelial cells. Scale bar, 20 μm. (E) Confocal image showing the cell tight junction (ZO-1 + ) between regenerated alveoli epithelial cells. Scale bar, 20 μm. (F) Direct fluorescence image of the transplanted GFP-labeled SOX9 + BCs (green) and bright-field image of tail vein delivered gold nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm. (G) Direct fluorescence image of the transplanted GFP-labeled SOX9 + BCs (green) and bright-field image of intratracheally delivered gold nanoparticles (AuNPs) of the same region (brown). Scale bar, 100 μm

    Article Snippet: Primary antibodies used in this work include BC markers:KRT5 (1:200, EP1601Y, Thermo), P63 (deltaN, 1:200, 4A4, Abcam), E-cadherin (1:200, H-108, Santa cruz), SOX9 (1:200, ERP14335-78, Abcam), SOX9 (1:200, AF3075, R & D); AEC markers: AQP5 (1:1000, EPR3747, Abcam), HOPX (1:500, E-1, Santa Cruz), PDPN (1:500, FL-162, Santa Cruz), SPC (1:200, M-20, Santa Cruz), SPC (1:200, FL-197, Santa Cruz), LAMP3 (1:200, 12632-1-AP, Proteintech); bronchiolar cell markers: CC10 (1:200, T-18, Santa Cruz), acetylated-α-Tubulin (1:1000, 6-11B-1, Abcam), MUC5AC (1:500, 45M1, Thermo), FOXJ1 (1:200, 2A5, eBioscience); vasculature markers: CD31 (1:100, M-20, Santa Cruz), CD34 (1:1000, EP373Y, Abcam); myofibroblast marker: α-SMA (1:500, 1A4, DAKO), Fibronectin (1:500, F14, Abcam), others: KI67 (1:200, RM-9106, Thermo), GFP (1:200, B-2, Santa Cruz), GFP (1:200, FL, Santa Cruz), GFP (1:200, T-19, Santa Cruz), ITGB1 (1:500, ERP16895, Abcam), Human specific Lamin A+C (1:200, EPR4100, Abcam).

    Techniques: Functional Assay, Marker, Immunofluorescence, Fluorescence, Labeling

    Degradation of Os WRKY11 protein by the UPS. a Rice ( Oryza sativa ) protoplasts were transformed with 35S:: OsWRKY11-YFP . After 4 h, protoplasts were treated with 100 μM MG132 and buffer for 1 day. Fluorescence was observed using a confocal laser scanning microscope. Scale bars: 30 μm. NT: non-treated control. b Immunoblot analysis of total protoplast proteins in the absence (−) or presence (+) of 100 μM MG132. Blots were probed with green fluorescent protein (GFP) antibody. The PAT antibody was used as a control for transformation efficiency

    Journal: Rice

    Article Title: Rice WRKY11 Plays a Role in Pathogen Defense and Drought Tolerance

    doi: 10.1186/s12284-018-0199-0

    Figure Lengend Snippet: Degradation of Os WRKY11 protein by the UPS. a Rice ( Oryza sativa ) protoplasts were transformed with 35S:: OsWRKY11-YFP . After 4 h, protoplasts were treated with 100 μM MG132 and buffer for 1 day. Fluorescence was observed using a confocal laser scanning microscope. Scale bars: 30 μm. NT: non-treated control. b Immunoblot analysis of total protoplast proteins in the absence (−) or presence (+) of 100 μM MG132. Blots were probed with green fluorescent protein (GFP) antibody. The PAT antibody was used as a control for transformation efficiency

    Article Snippet: Immunoblot analysis was performed with GFP antibody (Abcam; code: ab6556).

    Techniques: Transformation Assay, Fluorescence, Laser-Scanning Microscopy

    Membrane TK1 expression in of colon, breast, and lung cancer cell lines. Flow cytometry analysis of cell lines treated with anti-TK1 antibodies. a Quantification of TK1 expression on the cell membrane of HT-29 and SW620 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. b Quantification of TK1 expression on the cell membrane of MCF7 and MDA-MB-231 cell lines. The top bar graph shows MCF7 and MDA-MB-231 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. c Quantification of TK1 expression on the cell membrane of NCI-H460 and A549 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. Statistical analysis was performed by comparing the mouse isotype control fluorescent levels to those of A72, A74, CB1, or ab91651. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001; ns = P > 0.05

    Journal: Cancer Cell International

    Article Title: Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies

    doi: 10.1186/s12935-018-0633-9

    Figure Lengend Snippet: Membrane TK1 expression in of colon, breast, and lung cancer cell lines. Flow cytometry analysis of cell lines treated with anti-TK1 antibodies. a Quantification of TK1 expression on the cell membrane of HT-29 and SW620 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. b Quantification of TK1 expression on the cell membrane of MCF7 and MDA-MB-231 cell lines. The top bar graph shows MCF7 and MDA-MB-231 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. c Quantification of TK1 expression on the cell membrane of NCI-H460 and A549 cell lines stained with FITC or APC-conjugated anti-TK1 antibodies. Statistical analysis was performed by comparing the mouse isotype control fluorescent levels to those of A72, A74, CB1, or ab91651. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001; ns = P > 0.05

    Article Snippet: The commercially available antibody (ab91651) was conjugated to FITC or APC using a conjugation kit (EasyLink, Abcam, ab102884) and stored in the dark at 4 °C.

    Techniques: Expressing, Flow Cytometry, Cytometry, Staining, Multiple Displacement Amplification

    Subcellular localization of K3 is not different from wildtype AID. (A) Schematic representation of AID-GFP fusion constructs used for localization assays. GFP is fused to the C-terminus of AID or mutant K3. (B) HEK293T cells were transiently transfected with the respective GFP-fusion constructs. Two days post transfection, cells were treated as indicated with Leptomycin B (LMB) for 3 hours. Cells were fixed and localization of the respective GFP fusion proteins visualized using fluorescence microscopy. (C) The k46 B cell line stably expressing AID-GFP or K3-GFP fusion proteins were treated with or without Leptomycin B (LMB) for 3 hours and localization of GFP fusion constructs was determined using fluorescence microscopy.

    Journal: PLoS ONE

    Article Title: Lysine Residue at Position 22 of the AID Protein Regulates Its Class Switch Activity

    doi: 10.1371/journal.pone.0030667

    Figure Lengend Snippet: Subcellular localization of K3 is not different from wildtype AID. (A) Schematic representation of AID-GFP fusion constructs used for localization assays. GFP is fused to the C-terminus of AID or mutant K3. (B) HEK293T cells were transiently transfected with the respective GFP-fusion constructs. Two days post transfection, cells were treated as indicated with Leptomycin B (LMB) for 3 hours. Cells were fixed and localization of the respective GFP fusion proteins visualized using fluorescence microscopy. (C) The k46 B cell line stably expressing AID-GFP or K3-GFP fusion proteins were treated with or without Leptomycin B (LMB) for 3 hours and localization of GFP fusion constructs was determined using fluorescence microscopy.

    Article Snippet: Samples were blotted onto PVDF membranes (Millipore) and detected by Western blot analysis using polyclonal rabbit serum specific for human AID (Abcam), GFP (Cell Signaling), or actin (Cell Signaling) and HRP-conjugated secondary antibodies specific for rabbit IgG (Dako).

    Techniques: Construct, Mutagenesis, Transfection, Fluorescence, Microscopy, Stable Transfection, Expressing

    Somatic Hypermutation of IgV genes by wt AID and mutant K3. (A) Somatic hypermutation reflected as loss of IgM expression in AID −/− ΨV −/− IgM + DT40 cells. AID −/− ΨV −/− IgM + DT40 cells were stably transfected with plasmids encoding AID or K3 coupled to IRES-GFP expression. The percentage of sIgM-loss variants of GFP + cells is expressed as the median ± SE of multiple (n) independent clonal transfectants determined three weeks after transfection (untransfected control, n = 12; AID, n = 36; K3, n = 44). (B) Proportion of sequences carrying mutations in IgV genes of DT40 cells. IgV regions were cloned from cDNA of AID and K3 expressing DT40 transfectants. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of each chart. The frequency of mutations per bp sequenced and the total number of independent sequences analyzed is indicated underneath and in the center of each chart, respectively. (C) The abundance of AID and K3 in DT40 transfectants was determined by Western blotting from cell extracts. Blots were reprobed for GFP expression and actin levels as loading control. (D) Proportion of sequences carrying Bcl6 mutations in AID deficient B cells retrovirally transfected with AID or K3 constructs, respectively. Bcl6 was cloned from genomic DNA of sorted mouse B cells, retrovirally transfected to express AID or K3, respectively. Pie charts were generated as described in (B).

    Journal: PLoS ONE

    Article Title: Lysine Residue at Position 22 of the AID Protein Regulates Its Class Switch Activity

    doi: 10.1371/journal.pone.0030667

    Figure Lengend Snippet: Somatic Hypermutation of IgV genes by wt AID and mutant K3. (A) Somatic hypermutation reflected as loss of IgM expression in AID −/− ΨV −/− IgM + DT40 cells. AID −/− ΨV −/− IgM + DT40 cells were stably transfected with plasmids encoding AID or K3 coupled to IRES-GFP expression. The percentage of sIgM-loss variants of GFP + cells is expressed as the median ± SE of multiple (n) independent clonal transfectants determined three weeks after transfection (untransfected control, n = 12; AID, n = 36; K3, n = 44). (B) Proportion of sequences carrying mutations in IgV genes of DT40 cells. IgV regions were cloned from cDNA of AID and K3 expressing DT40 transfectants. Segment sizes in the pie charts are proportional to the number of sequences carrying the number of mutations indicated in the periphery of each chart. The frequency of mutations per bp sequenced and the total number of independent sequences analyzed is indicated underneath and in the center of each chart, respectively. (C) The abundance of AID and K3 in DT40 transfectants was determined by Western blotting from cell extracts. Blots were reprobed for GFP expression and actin levels as loading control. (D) Proportion of sequences carrying Bcl6 mutations in AID deficient B cells retrovirally transfected with AID or K3 constructs, respectively. Bcl6 was cloned from genomic DNA of sorted mouse B cells, retrovirally transfected to express AID or K3, respectively. Pie charts were generated as described in (B).

    Article Snippet: Samples were blotted onto PVDF membranes (Millipore) and detected by Western blot analysis using polyclonal rabbit serum specific for human AID (Abcam), GFP (Cell Signaling), or actin (Cell Signaling) and HRP-conjugated secondary antibodies specific for rabbit IgG (Dako).

    Techniques: Mutagenesis, Expressing, Stable Transfection, Transfection, Clone Assay, Western Blot, Construct, Generated

    Effect of lysine to arginine mutations in the AID protein on CSR activity. (A) Schematic representation of AID highlighting the position of the lysine (K) residues. For CSR assays, untagged constructs were expressed bicistronically together with GFP separated by an internal ribosome entry site (IRES). (B) Representative FACS profile for CSR activity of individual Lys to Arg mutants. B cells from AID deficient mice were retrovirally transfected with the respective lysine AID mutant (Mx: empty vector) and CSR to IgG1 was determined upon stimulation of cells with LPS and IL-4. GFP expression of infected cells was driven from an IRES element located 3′ of the stop codon of the cloned AID mutant. The percentage in the upper right quadrants indicate the percentage of IgG1 + cells among the GFP + (i.e. retrovirally infected) population. (C) Results from three independent experiments of a screen of lysine mutants showing the percentage of IgG1 + cells among the GFP + population. (D) Results from 11 independent experiments comparing the CSR activity of AID and K3. (E) Western Blot on lysates from B cells retrovirally transfected with the respective constructs. Black arrowheads indicate specific band. White arrowheads indicate nonspecific band. (IB: immune blot).

    Journal: PLoS ONE

    Article Title: Lysine Residue at Position 22 of the AID Protein Regulates Its Class Switch Activity

    doi: 10.1371/journal.pone.0030667

    Figure Lengend Snippet: Effect of lysine to arginine mutations in the AID protein on CSR activity. (A) Schematic representation of AID highlighting the position of the lysine (K) residues. For CSR assays, untagged constructs were expressed bicistronically together with GFP separated by an internal ribosome entry site (IRES). (B) Representative FACS profile for CSR activity of individual Lys to Arg mutants. B cells from AID deficient mice were retrovirally transfected with the respective lysine AID mutant (Mx: empty vector) and CSR to IgG1 was determined upon stimulation of cells with LPS and IL-4. GFP expression of infected cells was driven from an IRES element located 3′ of the stop codon of the cloned AID mutant. The percentage in the upper right quadrants indicate the percentage of IgG1 + cells among the GFP + (i.e. retrovirally infected) population. (C) Results from three independent experiments of a screen of lysine mutants showing the percentage of IgG1 + cells among the GFP + population. (D) Results from 11 independent experiments comparing the CSR activity of AID and K3. (E) Western Blot on lysates from B cells retrovirally transfected with the respective constructs. Black arrowheads indicate specific band. White arrowheads indicate nonspecific band. (IB: immune blot).

    Article Snippet: Samples were blotted onto PVDF membranes (Millipore) and detected by Western blot analysis using polyclonal rabbit serum specific for human AID (Abcam), GFP (Cell Signaling), or actin (Cell Signaling) and HRP-conjugated secondary antibodies specific for rabbit IgG (Dako).

    Techniques: Activity Assay, Construct, FACS, Mouse Assay, Transfection, Mutagenesis, Plasmid Preparation, Expressing, Infection, Clone Assay, Western Blot

    Hematopoietic cells in human placenta. Multiple clusters of CD34+cells, not associated with fetal or maternal circulation, are present in placental tissue. Significant numbers of CD34 cells are also positive for hematopoietic precursor markers such as CD90, CD38 and CD133. Paraffin sections of human placenta stained for CD34, CD90, CD38, and CD133 are shown. A1 : CD90 (green), A2 : CD34 (red), A3 : merged image of A1 and A2 with nuclear co-staining (DAPI blue), size indication bar is 10 µm. The white arrow points to CD34+CD90+cells. B1 : CD38 (green), B2 : CD34 (red), B3 : merged image of B1 and B2 with nuclear co-staining (DAPI blue), size indication bar is 100 µm. The white arrows point to CD34+ CD38+ cells. The green arrow points at a CD34+ capillary. C1 : CD133 (green), C2 : CD34 (red), C3 : merged image, showing staining for CD34 (red), CD133 (green), and nuclei (DAPI blue), size indication bar is 20 µm. The white arrows point to CD34+CD133+cells not associated with structures of the circulation (capillary walls). The green arrow points at the CD34+CD133+capillary. D : Negative control staining. D1 : merged image of staining with isotype anti-mouse antibody and isotype anti-rabbit antibody, secondary FITC (green) anti-mouse and Alexa Fluor 633 anti-rabbit antibody (red), co-stained for nuclei with DAPI (blue). No signals from cell markers other than nuclei are present. D2 : Merged image of staining with mouse anti-CD34 antibody and isotype anti-rabbit antibody, secondary FITC anti-mouse (green) and Alexa Fluor 633 anti-rabbit (red) antibody, co-stained for nuclei with DAPI (blue). Only signals from the CD34 cell marker and nuclei are present. D3 : Isotype anti-mouse antibody and anti-CD133 rabbit antibody, secondary FITC anti-mouse and Alexa Fluor 633 anti-rabbit antibody, co-staining DAPI (blue), merged image. Only a signal for CD133+cells is observed, no signals from CD34 cell markers are present. The size indication bar is 50 µm.

    Journal: Experimental Biology and Medicine (Maywood, N.j.)

    Article Title: Human Term Placenta as a Source of Hematopoietic Cells

    doi: 10.3181/0809-BC-262

    Figure Lengend Snippet: Hematopoietic cells in human placenta. Multiple clusters of CD34+cells, not associated with fetal or maternal circulation, are present in placental tissue. Significant numbers of CD34 cells are also positive for hematopoietic precursor markers such as CD90, CD38 and CD133. Paraffin sections of human placenta stained for CD34, CD90, CD38, and CD133 are shown. A1 : CD90 (green), A2 : CD34 (red), A3 : merged image of A1 and A2 with nuclear co-staining (DAPI blue), size indication bar is 10 µm. The white arrow points to CD34+CD90+cells. B1 : CD38 (green), B2 : CD34 (red), B3 : merged image of B1 and B2 with nuclear co-staining (DAPI blue), size indication bar is 100 µm. The white arrows point to CD34+ CD38+ cells. The green arrow points at a CD34+ capillary. C1 : CD133 (green), C2 : CD34 (red), C3 : merged image, showing staining for CD34 (red), CD133 (green), and nuclei (DAPI blue), size indication bar is 20 µm. The white arrows point to CD34+CD133+cells not associated with structures of the circulation (capillary walls). The green arrow points at the CD34+CD133+capillary. D : Negative control staining. D1 : merged image of staining with isotype anti-mouse antibody and isotype anti-rabbit antibody, secondary FITC (green) anti-mouse and Alexa Fluor 633 anti-rabbit antibody (red), co-stained for nuclei with DAPI (blue). No signals from cell markers other than nuclei are present. D2 : Merged image of staining with mouse anti-CD34 antibody and isotype anti-rabbit antibody, secondary FITC anti-mouse (green) and Alexa Fluor 633 anti-rabbit (red) antibody, co-stained for nuclei with DAPI (blue). Only signals from the CD34 cell marker and nuclei are present. D3 : Isotype anti-mouse antibody and anti-CD133 rabbit antibody, secondary FITC anti-mouse and Alexa Fluor 633 anti-rabbit antibody, co-staining DAPI (blue), merged image. Only a signal for CD133+cells is observed, no signals from CD34 cell markers are present. The size indication bar is 50 µm.

    Article Snippet: Slices were washed, incubated with blocking solution for 20 minutes and then incubated with secondary antibody (1:500) labeled with FITC- or Alexa Fluor-633 for 60 minutes at 37°C, washed, and mounted on slides with Gold Antifade reagent (Molecular Probes, Eugene, OR).

    Techniques: Staining, Negative Control, Marker

    β-catenin and TCF show enrichment when localized to the nucleus in successive cell cycles. A) Nuclear localization of Venus::SYS-1 in the ABpl lineage, shows the highest level in cells derived from consecutive posterior parents. B) Detail showing ABplaaa lineage; Black arrows show comparison between posterior cousin cells with high nuclear Venus::SYS-1 in two consecutive divisions (High-High or HH cells, ABplaaaapp marked “pp”), which have higher levels of nuclear Venus::SYS-1 than their cousins with parents that had no/low nuclear Venus::SYS-1 (Low-High or LH cells, ABplaaaaap marked “ap). C) Nuclear localization of GFP::POP-1, detectable after the 50 cell stage. GFP::POP-1 polarity is the opposite of Venus::SYS-1 with highest expression in cells derived from consecutive anterior parents. D) Detail of ABplppp lineage for GFP::POP-1 showing comparison between anterior cousin cells with high nuclear POP-1 in consecutive divisions (ABplpppaa marked “aa”) and their cousins with parents that had low nuclear POP-1 (ABplppppa marked “pa”). E) Venus::SYS-1 concentration for embryonic High-High cells (y axis) compared with their Low-High cousin cell. Most points fall above the red y = x line. F) Average GFP::POP-1 concentration for all High-High cells (y-axis) born after the 50 cell stage compared with their Low-High cousin cells. Most points fall above the red y = x line. G) Diagram illustrating comparison of SYS-1 and POP-1 asymmetry levels in daughter cells and graph showing POP-1 and SYS-1 asymmetry levels for all divisions after the 50-cell stage. Divisions of parent cells with high levels of POP-1 (blue) tend to have greater POP-1 asymmetry (x-axis) and less SYS-1 asymmetry (y-axis) than parent cells with high levels of SYS-1(red). H) Low/High labeling scheme and graph showing comparison of concentrations of Venus::SYS-1 and GFP::POP-1 for all individual cells after the 50-cell stage, colored by whether they are POP-1 High-High (dark blue), POP-1 Low-High (light blue), SYS-1 High-High (red) or SYS-1 Low-High (pink). The relative ratio of Venus::SYS-1 fluorescence intensity to GFP::POP-1 intensity for each group can be estimated from slope of a best-fit line (indicated above each line). Concentration values can be negative because nuclear intensity is subtracted from local background.

    Journal: PLoS Genetics

    Article Title: Quantitative Differences in Nuclear β-catenin and TCF Pattern Embryonic Cells in C. elegans

    doi: 10.1371/journal.pgen.1005585

    Figure Lengend Snippet: β-catenin and TCF show enrichment when localized to the nucleus in successive cell cycles. A) Nuclear localization of Venus::SYS-1 in the ABpl lineage, shows the highest level in cells derived from consecutive posterior parents. B) Detail showing ABplaaa lineage; Black arrows show comparison between posterior cousin cells with high nuclear Venus::SYS-1 in two consecutive divisions (High-High or HH cells, ABplaaaapp marked “pp”), which have higher levels of nuclear Venus::SYS-1 than their cousins with parents that had no/low nuclear Venus::SYS-1 (Low-High or LH cells, ABplaaaaap marked “ap). C) Nuclear localization of GFP::POP-1, detectable after the 50 cell stage. GFP::POP-1 polarity is the opposite of Venus::SYS-1 with highest expression in cells derived from consecutive anterior parents. D) Detail of ABplppp lineage for GFP::POP-1 showing comparison between anterior cousin cells with high nuclear POP-1 in consecutive divisions (ABplpppaa marked “aa”) and their cousins with parents that had low nuclear POP-1 (ABplppppa marked “pa”). E) Venus::SYS-1 concentration for embryonic High-High cells (y axis) compared with their Low-High cousin cell. Most points fall above the red y = x line. F) Average GFP::POP-1 concentration for all High-High cells (y-axis) born after the 50 cell stage compared with their Low-High cousin cells. Most points fall above the red y = x line. G) Diagram illustrating comparison of SYS-1 and POP-1 asymmetry levels in daughter cells and graph showing POP-1 and SYS-1 asymmetry levels for all divisions after the 50-cell stage. Divisions of parent cells with high levels of POP-1 (blue) tend to have greater POP-1 asymmetry (x-axis) and less SYS-1 asymmetry (y-axis) than parent cells with high levels of SYS-1(red). H) Low/High labeling scheme and graph showing comparison of concentrations of Venus::SYS-1 and GFP::POP-1 for all individual cells after the 50-cell stage, colored by whether they are POP-1 High-High (dark blue), POP-1 Low-High (light blue), SYS-1 High-High (red) or SYS-1 Low-High (pink). The relative ratio of Venus::SYS-1 fluorescence intensity to GFP::POP-1 intensity for each group can be estimated from slope of a best-fit line (indicated above each line). Concentration values can be negative because nuclear intensity is subtracted from local background.

    Article Snippet: Slides were then washed with PBS plus 0.1% Tween-20 and incubated with a blocking buffer containing 1% BSA and 0.1% sodium azide for 30 minutes at 4°C, then incubated with primary antibodies against GFP (ab290, Abcam, 1:2000) and POP-1 (mAbRL2, kind gift of Rueyling Lin, 1:25) diluted in blocking buffer overnight at 4°C in a humidified chamber.

    Techniques: Derivative Assay, Expressing, Concentration Assay, Labeling, Fluorescence

    Effect of marker nucleotide, the detection approach and dTTP on the signal intensity. The samples containing Lep cells accidentally infected with mycoplasma were fixed by formaldehyde, permeabilized by Triton X-100 and mycoplasmas’ DNA was detected using enzymatic detection with Pol I. ( a ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). ( b ) The enzymatic mixture contained fluorescein-dUTP. ( c ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized by streptavidin conjugated with FITC. ( d ) The enzymatic mixture contained biotin-dUTP and an equimolar amount of dTTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). The images were acquired for 11.44 ms. Scale bar = 10 µm.

    Journal: Cells

    Article Title: A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy

    doi: 10.3390/cells8121510

    Figure Lengend Snippet: Effect of marker nucleotide, the detection approach and dTTP on the signal intensity. The samples containing Lep cells accidentally infected with mycoplasma were fixed by formaldehyde, permeabilized by Triton X-100 and mycoplasmas’ DNA was detected using enzymatic detection with Pol I. ( a ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). ( b ) The enzymatic mixture contained fluorescein-dUTP. ( c ) The enzymatic mixture contained biotin-dUTP. Biotin-dUTP was visualized by streptavidin conjugated with FITC. ( d ) The enzymatic mixture contained biotin-dUTP and an equimolar amount of dTTP. Biotin-dUTP was visualized using a primary antibody (rabbit anti-biotin) followed by a secondary antibody (conjugated with Alexa Fluor 488). The images were acquired for 11.44 ms. Scale bar = 10 µm.

    Article Snippet: If the detection of biotin-dUTP was performed by fluorescently labeled streptavidin, the samples were incubated with streptavidin from Streptomyces avidinii conjugated with FITC (1:100, Sigma Aldrich, Prague, Czech Republic) diluted in the Tris-NaCl buffer for 30 min. After washing with the Tris-NaCl buffer, the samples were mounted in the mounting medium.

    Techniques: Marker, Infection, Mass Spectrometry

    Coincidence of subpleural lymphatic plexuses and leakage sites. Image orientation: caudal (left), rostral (right). ADN–VEGF-C mice were analyzed after 0.075 mg/mL doxycycline for 7 days. A: Left panel: Whole mount of dorsal surface of thorax of Prox1-GFP control mouse after horseradish peroxidase (HRP) staining for GFP. Right panel: Drawing of whole mount where green lines show GFP + lymphatic vessels [thoracic duct (TD), paravertebral lymphatic vessel (PVL), and lymphatic plexus (LP)]. Gray lines mark the location of ribs. B: Paravertebral lymphatic vessel ( arrows indicate valves) and connecting lymphatic ( arrowheads indicate valves) to subpleural lymphatic plexuses. C: Fluorescence stereomicroscopic image of the pleural surface of the chest wall of a 3-week-old Prox1-GFP mouse showing lymphatic vessels (green; arrowheads ) that connect the TD to PVLs. Some regions of the connecting lymphatics ( arrow ) are obscured by overlying tissue. D: Images of a region of chest wall showing sites of tracer leakage (red; left panel ), LYVE-1–HRP staining of lymphatic plexuses (brown; middle panel ), and an overlay of the two (white; LYVE-1–HRP; right panel ). E: Enlarged images of lymphatic plexuses (LYVE-1–HRP) in chest wall of single transgenic control mouse and ADN–VEGF-C mouse, the latter showing bud-like outgrowths ( arrowheads ). F: Regional area per lymphatic plexus in single-transgenic and ADN–VEGF-C mice. Each dot shows the area of one plexus. Red lines indicate plexus area means. n = 7 mice ( D ); n = 4 mice per group ( E and F ). ∗∗ P

    Journal: The American Journal of Pathology

    Article Title: Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations

    doi: 10.1016/j.ajpath.2017.05.009

    Figure Lengend Snippet: Coincidence of subpleural lymphatic plexuses and leakage sites. Image orientation: caudal (left), rostral (right). ADN–VEGF-C mice were analyzed after 0.075 mg/mL doxycycline for 7 days. A: Left panel: Whole mount of dorsal surface of thorax of Prox1-GFP control mouse after horseradish peroxidase (HRP) staining for GFP. Right panel: Drawing of whole mount where green lines show GFP + lymphatic vessels [thoracic duct (TD), paravertebral lymphatic vessel (PVL), and lymphatic plexus (LP)]. Gray lines mark the location of ribs. B: Paravertebral lymphatic vessel ( arrows indicate valves) and connecting lymphatic ( arrowheads indicate valves) to subpleural lymphatic plexuses. C: Fluorescence stereomicroscopic image of the pleural surface of the chest wall of a 3-week-old Prox1-GFP mouse showing lymphatic vessels (green; arrowheads ) that connect the TD to PVLs. Some regions of the connecting lymphatics ( arrow ) are obscured by overlying tissue. D: Images of a region of chest wall showing sites of tracer leakage (red; left panel ), LYVE-1–HRP staining of lymphatic plexuses (brown; middle panel ), and an overlay of the two (white; LYVE-1–HRP; right panel ). E: Enlarged images of lymphatic plexuses (LYVE-1–HRP) in chest wall of single transgenic control mouse and ADN–VEGF-C mouse, the latter showing bud-like outgrowths ( arrowheads ). F: Regional area per lymphatic plexus in single-transgenic and ADN–VEGF-C mice. Each dot shows the area of one plexus. Red lines indicate plexus area means. n = 7 mice ( D ); n = 4 mice per group ( E and F ). ∗∗ P

    Article Snippet: Lymphatics in Prox1-GFP mice were stained for GFP (Aves Labs, Inc., Tigard, OR; 1:1000, chicken polyclonal GFP-1020).

    Techniques: Mouse Assay, Staining, Fluorescence, Transgenic Assay

    Abnormalities of paravertebral lymphatics in ADN–VEGF-C mice. Paravertebral lymphatics [brown; GFP–horseradish peroxidase (HRP)] in Prox1-GFP control and ADN–VEGF-C/Prox1-GFP littermates after 0.075 mg/mL doxycycline for 7 days. A: Thoracic wall whole mounts showing valves ( arrows ) in both examples, but bud-like lymphatic outgrowths ( arrowheads ) only in ADN–VEGF-C/Prox1-GFP mouse. Image orientation: caudal (left), rostral (right). B–D: Paravertebral lymphatics vessel diameter ( B ), number of bud-like outgrowths ( C ), and valves per millimeter vessel length ( D ). Each dot is the value for one paravertebral lymphatic (two vessels per mouse; three mice per group). Red lines indicate group means. ∗∗ P

    Journal: The American Journal of Pathology

    Article Title: Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations

    doi: 10.1016/j.ajpath.2017.05.009

    Figure Lengend Snippet: Abnormalities of paravertebral lymphatics in ADN–VEGF-C mice. Paravertebral lymphatics [brown; GFP–horseradish peroxidase (HRP)] in Prox1-GFP control and ADN–VEGF-C/Prox1-GFP littermates after 0.075 mg/mL doxycycline for 7 days. A: Thoracic wall whole mounts showing valves ( arrows ) in both examples, but bud-like lymphatic outgrowths ( arrowheads ) only in ADN–VEGF-C/Prox1-GFP mouse. Image orientation: caudal (left), rostral (right). B–D: Paravertebral lymphatics vessel diameter ( B ), number of bud-like outgrowths ( C ), and valves per millimeter vessel length ( D ). Each dot is the value for one paravertebral lymphatic (two vessels per mouse; three mice per group). Red lines indicate group means. ∗∗ P

    Article Snippet: Lymphatics in Prox1-GFP mice were stained for GFP (Aves Labs, Inc., Tigard, OR; 1:1000, chicken polyclonal GFP-1020).

    Techniques: Mouse Assay

    Abnormal flow pattern of fluorescent tracer in ADN–VEGF-C mice. Fluorescence stereomicroscopic images of Prox1-GFP control mouse ( A ) and ADN–VEGF-C/Prox1-GFP mouse on doxycycline for 7 days ( B ) showing lymphatic vessels (green) 5 minutes after injection of rhodamine-labeled Ricinus communis agglutinin I (RCA I) lectin tracer (red) into a mesenteric lymph node. Image orientation: caudal (left), rostral (right). Arrows mark tracer in paravertebral lymphatic vessel. n = 5 mice ( A and B ). Scale bar = 1 mm ( A and B ).

    Journal: The American Journal of Pathology

    Article Title: Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations

    doi: 10.1016/j.ajpath.2017.05.009

    Figure Lengend Snippet: Abnormal flow pattern of fluorescent tracer in ADN–VEGF-C mice. Fluorescence stereomicroscopic images of Prox1-GFP control mouse ( A ) and ADN–VEGF-C/Prox1-GFP mouse on doxycycline for 7 days ( B ) showing lymphatic vessels (green) 5 minutes after injection of rhodamine-labeled Ricinus communis agglutinin I (RCA I) lectin tracer (red) into a mesenteric lymph node. Image orientation: caudal (left), rostral (right). Arrows mark tracer in paravertebral lymphatic vessel. n = 5 mice ( A and B ). Scale bar = 1 mm ( A and B ).

    Article Snippet: Lymphatics in Prox1-GFP mice were stained for GFP (Aves Labs, Inc., Tigard, OR; 1:1000, chicken polyclonal GFP-1020).

    Techniques: Flow Cytometry, Mouse Assay, Fluorescence, Injection, Labeling

    Pleural mesothelial stomata and exfoliation in ADN–VEGF-C mice. A and B: Scanning electron microscopic (SEM) images of pleural mesothelium of control mouse ( A ) and ADN–VEGF-C mouse on 0.075 mg/mL doxycycline for 7 days ( B ). A: Left panel: Cuboidal mesothelial surface of parietal pleura. Middle panel: Boxed area from left panel shown at higher magnification, indicating two lymphatic stomata ( arrows ). Right panel: Flat mesothelial surface of parietal pleura. No stomata are present in this region. B: ADN–VEGF-C mouse. Left panel: Surface of parietal pleura showing large region lacking mesothelial cells but revealing foramina in matrix ( arrowheads ). Right panel: Boxed area from left panel shown at higher magnification, indicating abnormal mesothelial cells surrounded by region of exfoliation. C: Amount of mesothelial exfoliation and corresponding volume of chyle in the thorax of ADN–VEGF-C mice on doxycycline for 0 to 7 days. D: Drawings of exfoliated regions in three ADN–VEGF-C mice. Green marks regions of exfoliation in only one of the three mice; yellow, regions of colocalization of exfoliation in two of the three mice; and red, colocalization in all three mice. E: Drawings of the location of lymphatic plexuses in three Prox1-GFP control mice, as in D . F: Comparison of amount of mesothelial exfoliation 24 hours after intrathoracic injection of phosphate-buffered saline (PBS) or chyle in wild-type mice. SEM image of exfoliated region after intrathoracic injection of chyle into a wild-type mouse ( arrows ). n = 2 mice per group ( C , chyle volume on day 7); n = 3 or 4 mice per group ( B and C , other groups); n = 3 mice per group ( F ). ∗ P

    Journal: The American Journal of Pathology

    Article Title: Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations

    doi: 10.1016/j.ajpath.2017.05.009

    Figure Lengend Snippet: Pleural mesothelial stomata and exfoliation in ADN–VEGF-C mice. A and B: Scanning electron microscopic (SEM) images of pleural mesothelium of control mouse ( A ) and ADN–VEGF-C mouse on 0.075 mg/mL doxycycline for 7 days ( B ). A: Left panel: Cuboidal mesothelial surface of parietal pleura. Middle panel: Boxed area from left panel shown at higher magnification, indicating two lymphatic stomata ( arrows ). Right panel: Flat mesothelial surface of parietal pleura. No stomata are present in this region. B: ADN–VEGF-C mouse. Left panel: Surface of parietal pleura showing large region lacking mesothelial cells but revealing foramina in matrix ( arrowheads ). Right panel: Boxed area from left panel shown at higher magnification, indicating abnormal mesothelial cells surrounded by region of exfoliation. C: Amount of mesothelial exfoliation and corresponding volume of chyle in the thorax of ADN–VEGF-C mice on doxycycline for 0 to 7 days. D: Drawings of exfoliated regions in three ADN–VEGF-C mice. Green marks regions of exfoliation in only one of the three mice; yellow, regions of colocalization of exfoliation in two of the three mice; and red, colocalization in all three mice. E: Drawings of the location of lymphatic plexuses in three Prox1-GFP control mice, as in D . F: Comparison of amount of mesothelial exfoliation 24 hours after intrathoracic injection of phosphate-buffered saline (PBS) or chyle in wild-type mice. SEM image of exfoliated region after intrathoracic injection of chyle into a wild-type mouse ( arrows ). n = 2 mice per group ( C , chyle volume on day 7); n = 3 or 4 mice per group ( B and C , other groups); n = 3 mice per group ( F ). ∗ P

    Article Snippet: Lymphatics in Prox1-GFP mice were stained for GFP (Aves Labs, Inc., Tigard, OR; 1:1000, chicken polyclonal GFP-1020).

    Techniques: Mouse Assay, Injection

    Immunocytochemical assessment of surface phenotypes for KCs and MNCs under the influence of M1 or M2 inducers. Fluorescent microscopy images display immunostaining (FITC, green); cell nuclei are counterstained with DAPI (blue); KCs: Kupffer cells; MNCs: macrophages of monocytic lineage; scale bar, 50 μ m.

    Journal: BioMed Research International

    Article Title: Activated Macrophages of Monocytic Origin Predominantly Express Proinflammatory Cytokine Genes, Whereas Kupffer Cells Predominantly Express Anti-Inflammatory Cytokine Genes

    doi: 10.1155/2019/3912142

    Figure Lengend Snippet: Immunocytochemical assessment of surface phenotypes for KCs and MNCs under the influence of M1 or M2 inducers. Fluorescent microscopy images display immunostaining (FITC, green); cell nuclei are counterstained with DAPI (blue); KCs: Kupffer cells; MNCs: macrophages of monocytic lineage; scale bar, 50 μ m.

    Article Snippet: Expression of distinctive proteins of the activated macrophages was evaluated using antibodies to CD68 (1:100, Abcam, UK), iNOs (1:100, Abcam), Arg1 (1:100, Abcam), and CD206 (1:100, Santa Cruz, USA), in combination with FITC-conjugated secondary antibodies (1:200, Abcam); the nuclei were counterstained with DAPI (Sigma-Aldrich).

    Techniques: Microscopy, Immunostaining

    Effect of GLP on radiation-induced inhibition of HepG2 cell growth. HepG2 cells were treated with GLP (10 µM) for 72 h and then with radiation (6 Gy) for 30 min. Treated cells were stained with (A) DAPI (blue) and (B) anti-γ-H2AX antibody (red). GLP, Ganoderma lucidum polysaccharide; DAPI, 4′, 6-diamidino-2-phenylindole; IR, irradiation; DMSO, dimethyl sulfoxide. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Ganoderma lucidum polysaccharide enhances radiosensitivity of hepatocellular carcinoma cell line HepG2 through Akt signaling pathway

    doi: 10.3892/etm.2017.5340

    Figure Lengend Snippet: Effect of GLP on radiation-induced inhibition of HepG2 cell growth. HepG2 cells were treated with GLP (10 µM) for 72 h and then with radiation (6 Gy) for 30 min. Treated cells were stained with (A) DAPI (blue) and (B) anti-γ-H2AX antibody (red). GLP, Ganoderma lucidum polysaccharide; DAPI, 4′, 6-diamidino-2-phenylindole; IR, irradiation; DMSO, dimethyl sulfoxide. *P

    Article Snippet: Then cells were incubated with γ-H2AX antibodies (1:1,000; ab2893; Abcam, Cambridge, MA, USA) overnight at 4°C, washed in PBST for 10 min three times and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:5,000; ab97050; Abcam) at 37°C for 2 h. The nuclei were visualized by staining with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich; Merck KGaA).

    Techniques: Inhibition, Staining, Irradiation

    Spermatocyte apoptosis in Brg1 KO testis. A ) Apoptotic cells were visualized using TUNEL assays detecting DNA breaks. B ) FACS analysis of apoptosis based on DNA content and cell morphology. Testis single cell suspension was stained with PI to reveal haploid

    Journal: Biology of Reproduction

    Article Title: Essential Roles of the Chromatin Remodeling Factor Brg1 in Spermatogenesis in Mice 1

    doi: 10.1095/biolreprod.111.097097

    Figure Lengend Snippet: Spermatocyte apoptosis in Brg1 KO testis. A ) Apoptotic cells were visualized using TUNEL assays detecting DNA breaks. B ) FACS analysis of apoptosis based on DNA content and cell morphology. Testis single cell suspension was stained with PI to reveal haploid

    Article Snippet: Apoptosis was analyzed on cryosections using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon International).

    Techniques: TUNEL Assay, FACS, Staining

    Association between gp78 and cidec in hepatic steatosis. (A) Nuclei were stained with DAPI, (a) gp78 was visualized (fluorescein isothiocyanate conjugated secondary antibody; red staining). (b) Cidec was visualized (tetraethyl rhodamine isothiocyanate conjugated secondary antibody; green staining); (c) Colocalization of gp78 and cidec (yellow) visualized on the surface of lipid droplets using immunofluorescent microscopy and identified by coimmunoprecipitation (scale bar=15 µm). (B) Gp78 and Cidec direct interaction detected by coimmunoprecipitation assay. gp78, glycoprotein; cidec, cell death-inducing DFFA-like effector c.

    Journal: Molecular Medicine Reports

    Article Title: Role of glycoprotein 78 and cidec in hepatic steatosis

    doi: 10.3892/mmr.2017.6834

    Figure Lengend Snippet: Association between gp78 and cidec in hepatic steatosis. (A) Nuclei were stained with DAPI, (a) gp78 was visualized (fluorescein isothiocyanate conjugated secondary antibody; red staining). (b) Cidec was visualized (tetraethyl rhodamine isothiocyanate conjugated secondary antibody; green staining); (c) Colocalization of gp78 and cidec (yellow) visualized on the surface of lipid droplets using immunofluorescent microscopy and identified by coimmunoprecipitation (scale bar=15 µm). (B) Gp78 and Cidec direct interaction detected by coimmunoprecipitation assay. gp78, glycoprotein; cidec, cell death-inducing DFFA-like effector c.

    Article Snippet: The cells were then incubated with primary antibodies specific for gp78 (ab54787; dilutions 1:1,000, Abcam) or cidec (ab213693; dilutions 1:1,000, Abcam) overnight at 4°C, followed by incubation with secondary antibodies conjugated with fluorescein isothiocyanate for gp78 (ab6785; dilutions 1:3,000, Abcam) and with tetraethyl rhodamine isothiocyanate for cidec (ab6718; 1:3,000, Abcam) for 1 h at 37°C.

    Techniques: Staining, Microscopy, Co-Immunoprecipitation Assay

    Co-localization of IRF3 and HSPD1. A. HeLa cells were transfected with the MAVS or control plasmid. At 8 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 and mouse antibody against HSPD1 and further incubated with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L (FITC). Nuclei were stained with DAPI. B. HeLa cells were transfected with the MAVS or control plasmid. At 16 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 (phospho S386) and mouse antibody against HSPD1 and further developed with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L (FITC). Nuclei were stained with DAPI.

    Journal: PLoS ONE

    Article Title: HSPD1 Interacts with IRF3 to Facilitate Interferon-Beta Induction

    doi: 10.1371/journal.pone.0114874

    Figure Lengend Snippet: Co-localization of IRF3 and HSPD1. A. HeLa cells were transfected with the MAVS or control plasmid. At 8 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 and mouse antibody against HSPD1 and further incubated with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L (FITC). Nuclei were stained with DAPI. B. HeLa cells were transfected with the MAVS or control plasmid. At 16 h post-transfection, the cells were fixed, permeabilized, and then stained with rabbit antibody against IRF3 (phospho S386) and mouse antibody against HSPD1 and further developed with goat anti-mouse IgG H L (Cy3) and goat anti-rabbit IgG H L (FITC). Nuclei were stained with DAPI.

    Article Snippet: After washing 3 times, the cells were stained with goat anti-mouse IgG H & L (Cy3) (Abcam) and goat anti-rabbit IgG H & L (FITC) (Abcam) for 45 min and then further stained with or without 4–6-diamidino-2-phenylindole-dihydrochloride (DAPI) (Invitrogen) for 15 min.

    Techniques: Transfection, Plasmid Preparation, Staining, Incubation