Figure 1.
The structure of biotinylated nucleoside ligands.
The picture shows the chemical structure of biotinylated BrdU, EdU and 2′-deoxythymidine ligands.
Figure 2.
The affinity of antibodies to BrdU, EdU and 2′-deoxythymidine on well plates and in cellular DNA.
A) A comparison of the relative fluorescence intensity after the reaction of ten primary antibodies with BrdU, EdU and 2′-deoxythymidine anchored to the streptavidin-coated well plates. B) The ratio between the EdU and BrdU signals for streptavidin-coated well plates. C) The ratio between the EdU and BrdU signals for nuclear DNA after a two-hour incorporation of BrdU or EdU. The signal strength was determined according to ([13]; light grey columns) or by the evaluation of the mean intensities of the signal of labelled nuclei (dark grey columns, see also Material and Methods). Besides the antibody clone MoBu-1, all the other antibodies reacted both with BrdU and EdU.
Figure 3.
The detection of EdU in fixed HeLa cells.
The picture shows examples of the detection of EdU in fixed and permeabilised cells with six antibodies after a ten-minute EdU labelling pulse. Note that only the clone MoBu-1 exhibits no signal. Barr: 20 µm.
Figure 4.
The detection of EdU after the click reaction with various fluorescent azido dyes. A.
The results of the detection of EdU using the antibody clone BU1/75 and anti-rat antibody conjugated with FITC (F) or Cy3 (C) after the click reaction with 0.2 mM fluorescent azido-dyes or without the click reaction (control) in fixed and permeabilised cells labelled for 10 minutes with EdU are shown. The cells were incubated with 4N HCl prior to the click reaction. The images were acquired for various times in order to demonstrate that the EdU-derived signal of the anti-BrdU antibody is not completely removed by the click reaction with an elevated concentration of azido-dyes. The images in the inserts were acquired for the same time (190 ms for cells labelled with anti-rat antibody FITC conjugate and 74 ms for cells labelled with anti-rat Cy3 conjugate). Their intensity therefore reflects the decrease of the EdU-derived signal of the anti-BrdU antibody signal after the click reaction with respect to the control cells. Barr: 20 µm. B. The level of the suppression of the signal is shown for the individual azido dyes as the ratio (R) between the time length necessary to achieve the first signs of saturation in the image of the evaluated sample and in the image of control sample without the click reaction (light grey columns). Alternatively, we used the ratio between the mean intensity of the images of nuclei of the control cells and sample cells (dark grey columns).
Figure 5.
The increase of azido dye concentration and acid treatment results in a non-specific signal.
The picture shows examples of the detection of incorporated EdU in DNA by means of a click reaction with 0.02 mM fluorescent dye without the HCl treatment (images labelled as control) and of detection with 0.2 mM fluorescent azido dyes and the antibody clone BU1/75 (Cy3 anti-rat and FITC anti-rat secondary antibodies) in cells treated with 4N HCl. Note that the higher concentration of fluorescent azido dyes and the subsequent treatment with 4N HCl led to a non-specific signal mainly in the nucleolus area. Barr: 20 µm.
Figure 6.
The suppression of the EdU signal using non-fluorescent azido molecules.
The suppression of the signal provided by BU1/75 antibody after a click reaction with 2 or 20 mM 2-azidoethanol, 1-azido-2,3-dihydroxypropane or azidomethylphenylsulfide in cells labelled with EdU for 20 minutes. The images were acquired for 8 ms. Barr: 20 µm.
Figure 7.
The simultaneous localisation of BrdU and EdU.
The results of simultaneous localisation of BrdU and EdU using two different protocols for BrdU revelation in cells labelled for 5 minutes with BrdU and then after 13 hours for 20 minutes with EdU. A. The revelation of BrdU by 4N HCl is shown in the picture. The upper set of images represents the detection of both signals without the application of the blocking step by means of 2 mM azidomethylphenylsulfide. The bottom set of images represents the detection of both signals with the application of the blocking step by means of 2 mM azidomethylphenylsulfide. Barr: 20 µm. B. The revelation of BrdU by copper(I) ions and exonuclease III is shown in the picture. The upper set of images represents the detection of both signals without the application of the blocking step by means of 2 mM azidomethylphenylsulfide. The bottom set of images represents the detection of both signals with the application of the blocking step by means of 2 mM azidomethylphenylsulfide. Barr: 20 µm.