gfp Affibody Search Results


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  • 99
    Lonza dulbecco s phosphate buffered saline dpbs
    Dulbecco S Phosphate Buffered Saline Dpbs, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dulbecco s phosphate buffered saline dpbs/product/Lonza
    Average 99 stars, based on 206 article reviews
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    dulbecco s phosphate buffered saline dpbs - by Bioz Stars, 2020-09
    99/100 stars
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    99
    Thermo Fisher phire hot start ii dna polymerase
    Phire Hot Start Ii Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 849 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 849 article reviews
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    phire hot start ii dna polymerase - by Bioz Stars, 2020-09
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    92
    Affibody cetuximab
    Examination of autofluorescence at both Affibdoy and <t>cetuximab</t> channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).
    Cetuximab, supplied by Affibody, used in various techniques. Bioz Stars score: 92/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cetuximab/product/Affibody
    Average 92 stars, based on 131 article reviews
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    cetuximab - by Bioz Stars, 2020-09
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    90
    Affibody sel tagged affibody molecules
    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting <t>Affibody</t> increasing as the tumors grow from time from inoculation
    Sel Tagged Affibody Molecules, supplied by Affibody, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sel tagged affibody molecules/product/Affibody
    Average 90 stars, based on 20 article reviews
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    sel tagged affibody molecules - by Bioz Stars, 2020-09
    90/100 stars
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    88
    Affibody affibody peptide
    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting <t>Affibody</t> increasing as the tumors grow from time from inoculation
    Affibody Peptide, supplied by Affibody, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    affibody peptide - by Bioz Stars, 2020-09
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    91
    Affibody angiopep 2
    Analysis of <t>angiopep-2</t> transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
    Angiopep 2, supplied by Affibody, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Affibody anti egfr targeted affibody
    Analysis of <t>angiopep-2</t> transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
    Anti Egfr Targeted Affibody, supplied by Affibody, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti egfr targeted affibody/product/Affibody
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    anti egfr targeted affibody - by Bioz Stars, 2020-09
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    99
    Thermo Fisher bacmam 2 0
    Analysis of <t>angiopep-2</t> transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
    Bacmam 2 0, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bacmam 2 0 - by Bioz Stars, 2020-09
    99/100 stars
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    99
    Thermo Fisher phusion hot start ii high fidelity dna polymerase
    Analysis of <t>angiopep-2</t> transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
    Phusion Hot Start Ii High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 2108 article reviews
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    phusion hot start ii high fidelity dna polymerase - by Bioz Stars, 2020-09
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    91
    Affibody unconjugated angiopep 2
    Analysis of <t>angiopep-2</t> transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
    Unconjugated Angiopep 2, supplied by Affibody, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    unconjugated angiopep 2 - by Bioz Stars, 2020-09
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    85
    Affibody affibody dependent rnai
    Analysis of <t>angiopep-2</t> transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
    Affibody Dependent Rnai, supplied by Affibody, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/affibody dependent rnai/product/Affibody
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    affibody dependent rnai - by Bioz Stars, 2020-09
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    88
    Affibody affibody zher2
    Analysis of <t>angiopep-2</t> transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.
    Affibody Zher2, supplied by Affibody, used in various techniques. Bioz Stars score: 88/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/affibody zher2/product/Affibody
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    affibody zher2 - by Bioz Stars, 2020-09
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    85
    Affibody alexa fluor 488 labelled affibody
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
    Alexa Fluor 488 Labelled Affibody, supplied by Affibody, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 488 labelled affibody/product/Affibody
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    alexa fluor 488 labelled affibody - by Bioz Stars, 2020-09
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    99
    Thermo Fisher alexafluor488 dye
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
    Alexafluor488 Dye, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Affibody anti fluorescein scfv
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
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    Affibody anti egfr affibody conjugated to alexa fluor 488
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different <t>anti-EGFR</t> Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
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    91
    Affibody two step procedure
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different <t>anti-EGFR</t> Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
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    Affibody based egfr targeted tracers
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different <t>anti-EGFR</t> Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
    Based Egfr Targeted Tracers, supplied by Affibody, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bl21
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different <t>anti-EGFR</t> Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
    Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher celllight actin gfp
    Effect of logD and charge on <t>affibody</t> conjugate mobility. Plots of mean instantaneous D fit for different <t>anti-EGFR</t> Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. <t>Alexa</t> 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.
    Celllight Actin Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Affibody biotin conjugated anti erbb2 affibody
    Correlative fluorescence microscopy and STEM of QD-labeled <t>ErbB2</t> on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.
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    Affibody plasma membrane
    Correlative fluorescence microscopy and STEM of QD-labeled <t>ErbB2</t> on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.
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    Millipore protease inhibitor cocktail
    Correlative fluorescence microscopy and STEM of QD-labeled <t>ErbB2</t> on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.
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    Affibody taq polymerase binding affibody molecule ztaq
    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z <t>Taq:3638</t> -ST-CH 3 . Comparison B illustrates uptake of the targeting <t>Affibody</t> increasing as the tumors grow from time from inoculation
    Taq Polymerase Binding Affibody Molecule Ztaq, supplied by Affibody, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).

    Journal: PLoS ONE

    Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

    doi: 10.1371/journal.pone.0060390

    Figure Lengend Snippet: Examination of autofluorescence at both Affibdoy and cetuximab channels over the tumor region Tumor outlined by GFP signal (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Area enclosed in the yellow box is shown at 20 times magnification in (C). Autofluorescence at both the cetuximab channel (D) and the Affibody channel (E) show significant contrast between tumor and non-tumor regions with autofluorescence greatest at the tumor center. No significant change between tumor interior, tumor edge and non-tumor area is seen for the fraction of signal at the Affibody channel (F).

    Article Snippet: The most striking observation of the Affibody and cetuximab maps is that on average cetuximab appears to be more confined to tumor interiors , while the Affibody appears more evenly dispersed throughout the tumor ( ).

    Techniques: Staining

    Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.

    Journal: PLoS ONE

    Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

    doi: 10.1371/journal.pone.0060390

    Figure Lengend Snippet: Comparison of raw fluorescent signals. Raw fluorescent signals from various regions shown at both cetuximab channel (left) and Affibody channel (right) using box and whisker plots. Signal from injected animals are offset to the left while those from non-injected control animals are offset to the right with boxes shaded. The central lines are the medians, the edges of the boxes are the 25th and 75th percentiles and individual data points are plotted as open circles.

    Article Snippet: The most striking observation of the Affibody and cetuximab maps is that on average cetuximab appears to be more confined to tumor interiors , while the Affibody appears more evenly dispersed throughout the tumor ( ).

    Techniques: Whisker Assay, Injection

    Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.

    Journal: PLoS ONE

    Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

    doi: 10.1371/journal.pone.0060390

    Figure Lengend Snippet: Examination of Affibody and cetuximab distribution over the tumor region. Signal from GFP outlines the tumor (A). H E stain of the same tissue slice showing the structural differences between the tumor area and adjacent normal tissue at 8 times magnification (B). Tumor is outlined in red and area enclosed in the yellow box is shown at 20 times magnification in (C). Fluorescent signal at cetuximab channel shows significant contrast in much of the tumor, but appears reduced around the edges (D). Fluorescent signal at Affibody channel shows significant contrast in the tumor and over a broader region of the tumor (E). Fraction of signal from the Affibody channel is shown in (F) and demonstrates significant deviation in signal from the two channels at the edge and interior of the tumor.

    Article Snippet: The most striking observation of the Affibody and cetuximab maps is that on average cetuximab appears to be more confined to tumor interiors , while the Affibody appears more evenly dispersed throughout the tumor ( ).

    Techniques: Staining

    Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.

    Journal: PLoS ONE

    Article Title: Fluorescent Affibody Peptide Penetration in Glioma Margin Is Superior to Full Antibody

    doi: 10.1371/journal.pone.0060390

    Figure Lengend Snippet: Comparison of plasma excretion for the two proteins. Plasma excretion data with error bars and bi-exponential curve fits to the data are shown for cetuximab-IRDye 680RD (left) and Affibody-IRDye 800CW (right). Curve fit equations are also shown where FL is fluorescence intensity. R-squared values of 0.71 and 0.90 for cetuximab and Affibody fits respectively.

    Article Snippet: The most striking observation of the Affibody and cetuximab maps is that on average cetuximab appears to be more confined to tumor interiors , while the Affibody appears more evenly dispersed throughout the tumor ( ).

    Techniques: Fluorescence

    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Article Snippet: Preparation and labeling of the Sel-tagged Affibody molecules The Sel-tagged Affibody molecules were successfully obtained by expression in E. coli as C-terminal fusions to green fluorescent protein (GFP), then recovered with immobilized metal ion affinity chromatography (IMAC), released by tobacco etch virus (TEV)-protease cleavage, and purified by high-performance liquid chromatography (HPLC).

    Techniques: Positron Emission Tomography

    PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Article Snippet: Preparation and labeling of the Sel-tagged Affibody molecules The Sel-tagged Affibody molecules were successfully obtained by expression in E. coli as C-terminal fusions to green fluorescent protein (GFP), then recovered with immobilized metal ion affinity chromatography (IMAC), released by tobacco etch virus (TEV)-protease cleavage, and purified by high-performance liquid chromatography (HPLC).

    Techniques: Positron Emission Tomography

    Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

    Journal: Nature Communications

    Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

    doi: 10.1038/ncomms15623

    Figure Lengend Snippet: Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

    Article Snippet: Angiopep-2 conjugated to the Bim BH3 peptide analogue had similar influx rate as unconjugated angiopep-2 , while the level of influx of angiopep-2 conjugated to an affibody was slightly lower than unconjugated angiopep-2, though significantly higher than that of the control affibody ( ).

    Techniques: Fluorescence, Expressing, Staining, Incubation, Concentration Assay, Confocal Microscopy, Permeability, Injection, Mouse Assay

    Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

    Journal: Nature Communications

    Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

    doi: 10.1038/ncomms15623

    Figure Lengend Snippet: Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

    Article Snippet: Angiopep-2 conjugated to the Bim BH3 peptide analogue had similar influx rate as unconjugated angiopep-2 , while the level of influx of angiopep-2 conjugated to an affibody was slightly lower than unconjugated angiopep-2, though significantly higher than that of the control affibody ( ).

    Techniques: In Vitro, Permeability, Co-Culture Assay, Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Microscopy, Expressing

    Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

    Journal: Nature Communications

    Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

    doi: 10.1038/ncomms15623

    Figure Lengend Snippet: Analysis of angiopep-2 transport in BBB spheroid. ( a ) Fluorescence images showing LRP-1 receptor expression (green) in spheroids established with primary HBMECs (pre-labelled in CellTracker Orange (shown in red)). Scale bar, 50 μm. ( b ) Fluorescence images showing the LRP-1 receptor expression (red) in immortalized hCMEC/D3 ECs. Nuclei of spheroids were stained with Hoechst dye (blue). Scale bar: 100 μm. ( c ) Confocal fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; compared to a corresponding scrambled peptide) in spheroids established with primary HBMECs. Spheroids were incubated with either angiopep-2 or scrambled-Cy5 peptide (10 μM) at 37 °C for 3 h. Scale bar, 100 μm. ( d ) Bar graph quantifying the transport of angiopep-2 (or scrambled peptide) at a concentration of 5 and 10 μM in spheroids established with primary HBMECs. Statistical analyses were performed using the one-way ANOVA and Bonferroni's multiple comparison test. ( e ) Fluorescence images showing the transport of Cy5-labelled angiopep-2 (cyan; conducted as in c ) in spheroids established with immortalized hCMEC/d3 cells. Scale bar, 100 μm. ( f ) Bar graph quantifying the transport of angiopep-2 (10 μM; from e ). Statistical analyses were performed using the Student's t -test. ( g ) Fluorescence images acquired using confocal microscopy showing the transport of Cy5-labelled angiopep-2 (cyan; 10 μM) in spheroids established with primary HBMECs at either 4 °C (to inhibit endo/transcytosis) or 37 °C for 3 h. Scale bar, 200 μm. ( h ) Bar graph quantifying the transport of angiopep-2 at either 4 or 37 °C (from g ). Statistical analyses were performed using the one-way ANOVA and Tukey's multiple comparison test. All graphs above depict mean Cy5 intensity quantified at 88 μm depth from the surface of the spheroid with s.d. error bars ( n spheroid =10, n experiment =3). ( i ) Co-incubation of spheroids with TAMRA-labelled angiopep-2 or angio-scramble (at 10 μM) and with FITC-dextran (70 kDa; at 10 μg ml −1 ) for 3 h. The graph displays the mean fluorescence intensity of the peptides (TAMRA) on the left y axis, and dextran (FITC) on the right y axis at 88 μm depth from the surface of each spheroid with s.d. error bars ( n spheroid =3–6, n experiment =3). Incubation of spheroids with each peptide did not increase spheroid permeability to FITC-dextran. Statistical analyses were performed using the two-way ANOVA and Dunnett's multiple comparison test. ( j ) Fluorescence images of brain cryosections showing the accumulation of angiopep-2 (red) in the brain tissue compared to the scrambled peptide. Angiopep-2 (or the scrambled peptide; 100 μg) were injected via the tail vein. Mice were killed after 24 h, and the brains were excised. The vasculature was stained with DyLight 488 lectin (green), while cell nuclei were labelled with Hoechst dye (blue). Scale bar, 50 μm.

    Article Snippet: Angiopep-2 conjugated to the Bim BH3 peptide analogue had similar influx rate as unconjugated angiopep-2 , while the level of influx of angiopep-2 conjugated to an affibody was slightly lower than unconjugated angiopep-2, though significantly higher than that of the control affibody ( ).

    Techniques: Fluorescence, Expressing, Staining, Incubation, Concentration Assay, Confocal Microscopy, Permeability, Injection, Mouse Assay

    Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

    Journal: Nature Communications

    Article Title: Blood-brain-barrier spheroids as an in vitro screening platform for brain-penetrating agents

    doi: 10.1038/ncomms15623

    Figure Lengend Snippet: Analysis of angiopep-2 transport using the well-established in vitro BBB Transwell system. Permeability assay using the BBB co-culture Transwell model showing that the ( a ) scrambled control and ( b ) angiopep-2 displayed significantly lower permeation in the co-culture model compared to inserts containing no cells (which represent passive diffusion). ( c ) The Transwell co-culture model failed to differentiate between the permeability of angiopep-2 and the scrambled peptide. For all permeability assays, TAMRA-labelled angiopep-2 (or scramble) peptide (10 μM concentration) was added onto the apical side of the Transwells of the co-culture model after 84 h of incubation. The basal side of the Transwell was imaged using fluorescence microscopy, and the fluorescence intensity was quantified over 40 h. The plots show the accumulation of fluorescence intensity over time with s.d. error bars ( n transwell =2, n experiment =2). Statistical analysis was performed using the one-way ANOVA and Tukey's multiple comparison test. ( d ) Confocal images showing higher expression of ZO-1 (tight junction), P-gp (efflux pump) and β-catenin (adherens junction; shown in white) on the surface of BBB spheroids compared with hCMEC/D3 ECs in the triple co-culture Transwell model after 48 h. Cell nuclei were labelled with Hoechst dye (shown in blue). Scale bar, 100 μm.

    Article Snippet: Angiopep-2 conjugated to the Bim BH3 peptide analogue had similar influx rate as unconjugated angiopep-2 , while the level of influx of angiopep-2 conjugated to an affibody was slightly lower than unconjugated angiopep-2, though significantly higher than that of the control affibody ( ).

    Techniques: In Vitro, Permeability, Co-Culture Assay, Diffusion-based Assay, Concentration Assay, Incubation, Fluorescence, Microscopy, Expressing

    Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Article Snippet: We have now investigated the mobility of a range of dye-EGFR affibody conjugates on T47D cells, using the value of D calculated for Alexa Fluor 488-labelled affibody as a reference.

    Techniques:

    Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Journal: PLoS ONE

    Article Title: Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

    doi: 10.1371/journal.pone.0074200

    Figure Lengend Snippet: Effect of logD and charge on affibody conjugate mobility. Plots of mean instantaneous D fit for different anti-EGFR Affibody conjugates vs charge at pH 7.4 ( A ), and logD ( B ). C) Plot of spot density for selected anti-EGFR Affibody conjugates vs charge at logD. Each datapoint corresponds to mean ± SEM of at least 10 independent areas. Lines show linear regression fit to the data, R 2 values indicating goodness of fit. Alexa 555 is not included in this figure as the structure is not published and charge and logD values are unavailable.

    Article Snippet: In our previous work we used mean instantaneous diffusion coefficient (D ) values as a measure of the mobility of dye conjugates bound to receptors in the plasma membrane of T47D cells, and showed that anti-EGFR affibody conjugated to Alexa Fluor 488 exhibits similar levels of mobility when bound to EGFR in cells to endogenously labelled EGFR-GFP.

    Techniques:

    Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.

    Journal: bioRxiv

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.1101/2020.01.14.906040

    Figure Lengend Snippet: Correlative fluorescence microscopy and STEM of QD-labeled ErbB2 on whole breast cancer cells revealing linear QD-chains in actin rich region. (A) Cropped fluorescence micrograph of a SKBR3 breast cancer cell showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. (B) Corresponding STEM micrograph acquired at the same spot ( M = 1,000×). (C) STEM micrograph of region marked in A. B at M =30,000×. (D) Region selected from C imaged at M =100,000 ×. Automatically detected QD-labels are marked in yellow to enhance the visibility. The white dotted line marks a linear QD-chain consisting of six QD-labels with an inter-label distance of 36 nm ± 15 nm and angle of 180° ± 30°.

    Article Snippet: In the following steps, the breast cancer cells were incubated with a specific biotin-conjugated anti-ErbB2-Affibody, fixated with paraformaldehyde to prevent artificial clustering of labels, and marked with fluorescent, streptavidin-conjugated QDs.

    Techniques: Fluorescence, Microscopy, Labeling

    Graphs of the pair correlation function g ( r ) versus the radial distance r between QD-labels, collected at actin-rich- and actin-low cell regions with correlating schemes. (A) g ( r ) of QD-labels detected in actin-rich cellular regions collected from 27 images of 11 cells. The red dotted line marks g ( r ) = 20 nm. As comparison, g ( r ) of simulated data is included of randomly positioned labels with the same particle density (see Table 1 ). (B) g ( r ) of QD-labels in actin-low regions collected in 17 images of 8 cells. The arrow marks a peak at r = 36 nm. The red dotted line is at r = 20 nm. g ( r ) of simulated data of randomly positioned labels is also included. (C) Schematic representation of the assumed distribution of ErbB2 and EGFR homo- and heterodimers in relation to cortical actin filaments and lipid rafts in actin-rich, ruffled cell regions. EGFR is bound to actin filaments while ErbB2 can move freely in the membrane and lipid rafts. Those ErbB2 molecules assembled in ErbB2:EGFR heterodimers are bound to helical actin filaments with a pitch of 36 nm. The number of ErbB2 homodimers in ruffled regions outweighs the number of postulated heterodimers. ( D ) Actin-low, flat cell regions with fewer ErbB2 homodimers being present than in ruffled regions, and postulated heterodimers dominating the analysis. The ruffled regions contain a higher number of growth factor receptors than the flat regions.

    Journal: bioRxiv

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.1101/2020.01.14.906040

    Figure Lengend Snippet: Graphs of the pair correlation function g ( r ) versus the radial distance r between QD-labels, collected at actin-rich- and actin-low cell regions with correlating schemes. (A) g ( r ) of QD-labels detected in actin-rich cellular regions collected from 27 images of 11 cells. The red dotted line marks g ( r ) = 20 nm. As comparison, g ( r ) of simulated data is included of randomly positioned labels with the same particle density (see Table 1 ). (B) g ( r ) of QD-labels in actin-low regions collected in 17 images of 8 cells. The arrow marks a peak at r = 36 nm. The red dotted line is at r = 20 nm. g ( r ) of simulated data of randomly positioned labels is also included. (C) Schematic representation of the assumed distribution of ErbB2 and EGFR homo- and heterodimers in relation to cortical actin filaments and lipid rafts in actin-rich, ruffled cell regions. EGFR is bound to actin filaments while ErbB2 can move freely in the membrane and lipid rafts. Those ErbB2 molecules assembled in ErbB2:EGFR heterodimers are bound to helical actin filaments with a pitch of 36 nm. The number of ErbB2 homodimers in ruffled regions outweighs the number of postulated heterodimers. ( D ) Actin-low, flat cell regions with fewer ErbB2 homodimers being present than in ruffled regions, and postulated heterodimers dominating the analysis. The ruffled regions contain a higher number of growth factor receptors than the flat regions.

    Article Snippet: In the following steps, the breast cancer cells were incubated with a specific biotin-conjugated anti-ErbB2-Affibody, fixated with paraformaldehyde to prevent artificial clustering of labels, and marked with fluorescent, streptavidin-conjugated QDs.

    Techniques:

    Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

    Journal: bioRxiv

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.1101/2020.01.14.906040

    Figure Lengend Snippet: Schematics of whole cell preparation for the analysis of membrane proteins. (A) Transduction of SKBR3 cells, seeded on silicon microchips with a silicon nitride membrane, with actin-green fluorescent protein (GFP)-constructs and cultivation of cells for 40 h. ( B ) Cells remain untreated (Control, left) or are treated with Cytochalasin (Cyt) D (right). (C) Membranous ErbB2 is labeled with streptavidin coated quantum dots (QDs) via Affibody-biotin conjugates. The cells are covered with graphene to protect against evaporation of the liquid in the vacuum of the electron microscope. ( D ) Configuration for fluorescence microscopy (FM, left), and for scanning transmission electron microscopy (STEM, right).

    Article Snippet: In the following steps, the breast cancer cells were incubated with a specific biotin-conjugated anti-ErbB2-Affibody, fixated with paraformaldehyde to prevent artificial clustering of labels, and marked with fluorescent, streptavidin-conjugated QDs.

    Techniques: Transduction, Construct, Labeling, Evaporation, Microscopy, Fluorescence, Transmission Assay, Electron Microscopy

    Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.

    Journal: bioRxiv

    Article Title: Correlative Fluorescence- and Electron Microscopy of Whole Breast Cancer Cells Reveals Different Distribution of ErbB2 Dependent on Underlying Actin

    doi: 10.1101/2020.01.14.906040

    Figure Lengend Snippet: Correlative fluorescence microscopy and STEM of whole breast cancer cells. (A, C) Cropped fluorescence micrographs of SKBR3 breast cancer cells showing cellular actin-GFP in green and QD-labeled membrane ErbB2 in red. Areas where both signals overlap appear yellow. Actin-containing ruffles can be identified as bright green and yellowish lines at the cell edges. (B, D) Corresponding STEM micrographs of graphene-covered breast cancer cells acquired at the same spots. B: Magnification M = 1,000×, D: M = 4,000×. (E, F) STEM micrographs of regions marked in C, and D acquired at higher magnification than in D. QD-labels appear as white dots. Membrane ruffle in E appears in a light grey shade compared to flat cell regions in F, appearing in a darker grey, E: M = 100,000×, F: M = 120,000×.

    Article Snippet: In the following steps, the breast cancer cells were incubated with a specific biotin-conjugated anti-ErbB2-Affibody, fixated with paraformaldehyde to prevent artificial clustering of labels, and marked with fluorescent, streptavidin-conjugated QDs.

    Techniques: Fluorescence, Microscopy, Labeling

    PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a Balbc nu/nu mouse (prone) bearing tumors ( white arrows ): a one A431 xenograft (1 × 10 7 cells, 15 days) or b two A431 xenografts ( left : 1 × 10 7 cells, 28 days; right : 1 × 10 7 cells, 25 days). Comparison A shows a 7-times higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 compared to the non-targeting [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates uptake of the targeting Affibody increasing as the tumors grow from time from inoculation

    Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Positron Emission Tomography

    PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Journal: EJNMMI Research

    Article Title: Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake

    doi: 10.1186/s13550-016-0213-8

    Figure Lengend Snippet: PET images, summed 30–60 min, and TACs from a SCID mouse (prone) bearing tumors ( white arrows ): a one FaDu xenograft (1 × 10 6 cells, 12 days) or b two FaDu xenografts ( left : (1 × 10 6 cells, 12 days); right : (0.5 × 10 6 cells, 12 days). Comparison A illustrates the higher uptake with targeting [methyl- 11 C]-Z EGFR:2377 -ST-CH 3 but with a ≈60 % non-targeting uptake of [methyl- 11 C]-Z Taq:3638 -ST-CH 3 . Comparison B illustrates the visually discernable heterogeneous uptake of the targeting Affibody in the larger tumor on the left. SUV mean is affected by whether the entire (1) or only central ROI (2) of the left tumor is used

    Article Snippet: DNA constructions and expression of Sel-tagged Affibody molecules The EGFR-binding Affibody molecule ZEGFR:2377 [ , ] and the irrelevant Taq polymerase-binding Affibody molecule ZTaq:3638 [ ] were fused with a C-terminal ST as previously described [ , ].

    Techniques: Positron Emission Tomography