36 research outputs found

    Direct comparison between aptamer and antibody stainings.

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    <p><b>(A)</b> Antibody concentration was first titrated to perform optimal live-stained as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173050#pone.0173050.g001" target="_blank">Fig 1A</a> (Ab1: red trace and Ab2: black trace). Every point in the saturation curves represents the average intensity from 20 to 50 cells imaged with an epifluorescence microscope from at least 3 independent experiments (associated error is displayed as the SEM). All further antibody stainings through out the manuscript were used at 1% v/v (from the original stock concentration provided by the supplier). (<b>B</b>) Equally scaled images to allow a direct comparison between the staining pattern, performance and fluorescence intensity of cells stained with aptamers or antibodies. Scale bar: 5 μm. The bar graphs at the right of each panel show the average intensity of cells stained with antibodies (Ab1 and Ab2) expressed in percentage of the aptamers intensities. Error bars are the SEM from 3–4 independent experiments with more than 30 cells per experiment. Detailed information of all Ab1 and Ab2 antibodies can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173050#pone.0173050.t001" target="_blank">Table 1</a> in the Materials and Methods section.</p

    Recognition of subcellular structures using STED microscopy in aptamer or antibody stained samples against Epha2.

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    <p>Figure legend is equivalent to legend on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173050#pone.0173050.g006" target="_blank">Fig 6</a>, but for Epha2.</p

    Co-localization of aptamers and classical endocytotic markers.

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    <p>(<b>A-C</b>) Confocal images of cells co-stained with aptamers and Alexa488-dextran (Dex) or Alexa488-Transferrin (Tf). (<b>D</b>) Confocal images of a validated aptamer against the transferrin receptor (TfR) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173050#pone.0173050.ref007" target="_blank">7</a>] on HEK293 cells was used as positive co-localization control with Tf. Pearson’s correlations coefficient (r) were calculated from 3 independent experiments (10 to 20 cells analyzed per experiments). The negative control (Neg.) was obtained by flipping one of the images horizontally before computing the correlation coefficient. Bars represent the average of three independent experiments and error is the SEM. Scale bar represents 5 μm.</p

    Live staining and aptamer specificity.

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    <p>(<b>A</b>) Simple scheme describing the procedure to stain living cells using aptamers or antibodies (probes). Cells are incubated with aptamer or antibodies allowing the internalization of the probes by the endocytosis of the receptor. (<b>B</b>) Aptamers against EGFR, ErbB2 and Epha2 were used to live-stain human cell lines expressing these receptors (A431, SKB3 and HeLa cells respectively). A control aptamer with randomized sequence of equivalent length and coupled to the same fluorophore was used to stain these cell lines. Additionally, cell lines that do not express such receptors were used as negative control cells (MCF7 cells are negative for EGFR and ErbB2; HEK293 cells are negative for Epha2). Both controls, the cell lines not expressing the receptor and the randomized aptamer show virtually no aptamer signal (nuclei were DAPI stained, displayed in blue). All images were acquired using an epifluorescence microscope with the same settings and they are equally scaled to allow a direct comparison. Scale bar represents 5 μm.</p

    Average intensity of single antibodies and aptamers.

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    <p><b>(A-C)</b> Examples of comparable STED images with single aptamers or single antibody packages (primary-secondary) seeded on glass coverslips. Scale bar (top image on panel A) represents 500 nm. The plots associated display the average intensity from more than thousand spots of single antibody packages expressed as fold of single aptamer intensity. Error bars are the SEM of 3 independent experiments.</p

    Estimations of the ability of antibodies or aptamers to recognized their epitopes.

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    <p><b>(A)</b> Scheme explaining how the epitope recognition estimation was calculated. (I) Cellular intensity (% of aptamer) is the data also displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173050#pone.0173050.g003" target="_blank">Fig 3B</a>. (II) Intensity of single antibody package expressed as fold over the aptamer intensity is the data on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173050#pone.0173050.g004" target="_blank">Fig 4A–4C</a>. (III) If we know the signal contribution of a single antibody package to the total cellular staining, we can estimate how many epitopes were found by the antibodies in relation to the epitopes found by the aptamers. (<b>B</b>) The results of the equation (III). Error bars represent the SEM from at least 3 independent experiments.</p

    Endogenous SNAP25 and SNAP25(1100) have a similar cellular distribution within SNAP25(1100) transfected PC12 cells.

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    (A) Visualization of both endogenous SNAP25 and SNAP25(1100) using SNAP25 specific primary and secondary antibodies, plus NbALFA. (B, C) Negative control experiments, leaving out either primary antibodies (B) or NbALFA (C). (D) Imaging control, using a mixture of the same secondary antibody with 2 distinct fluorophores (targeting both endogenous SNAP25 and SNAP25(1100)), to provide a visual indication of the maximum expected colocalization. Bottom part of the figure: legend for used symbols and schemes. Scale bars: 2.5 μm. For quantification, see S17 Fig. (TIF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded syntaxin 13.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 14C_STX13,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (TIF)</p

    Visualization of 15 nanobarcode epitopes using 4 spectrally distinct nanobodies.

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    (A-D) Nanobody-based identification of the 4 genetically encoded nanobarcode epitopes mCherry(Y71L), GFP(Y66L), syn87, and syn2 and the ALFA-tag epitope by their corresponding nanobodies NbRFP, NbEGFP, NbSyn87, NbSyn2, and NbALFA. Scaling was optimized for each protein. (D) VAMP4(1111) example (all epitopes present) and negative control condition: mock transfection (no DNA, no epitopes present) using same intensity scale. (E) As in (D), now with upscale intensities. Scale bar: 50 μm. (TIFF)</p
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