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FluoTag®-X4 anti-GFP single domain antibody shows a high correlation with the native GFP signal and is superior to conventional GFP antibodies in quantitative colocalization

 
FluoTag®-X4 anti-GFP-N0304-At542, GFP single domain antibody

The FluoTag®-X4 anti-GFP-N0304-At542 antibody is a mixture of two single domain antibodies with high affinity and specificity against two distinct epitopes of the GFP protein. Each antibody is directly labeled with two ATTO-542 fluorophore molecules (At542) to ensure bright signal. Single domain antibodies bind to their target in a monovalent way and are 10-fold smaller compared to conventional full IgG antibodies. Taken together, this leads to increased tissue penetrance and superior quantitative imaging.

The FluoTag®-X4 anti-GFP-N0304-At542 antibody is a mixture of two single domain antibodies with high affinity and specificity against two distinct epitopes of the GFP protein. Each antibody is directly labeled with two ATTO-542 fluorophore molecules (At542) to ensure bright signal. Single domain antibodies bind to their target in a monovalent way and are 10-fold smaller compared to conventional full IgG antibodies. Taken together, this leads to increased tissue penetrance and superior quantitative imaging.

In the following approach this advantage should be pointed out by a colocalization study of the native GFP signal with the signal of the antibody staining. Brain sections of transgenic microglia CX3CR1-GFP+/- mice (Jung et al., 2000) were immunohistochemically stained with either the FluoTag®-X4 anti-GFP antibody or by conventional indirect immunostaining.

Colocalization analyzed by merged pictures, scatter plots and correlation coefficient

Colocalization can be visualized in a merged picture of the two fluorescent signals. Overlapping pixels of the native GFP channel (green – figure 1A, 2A) and the channel of the antibody staining (red – figure 1B, 2B) appear in yellow (figure 1C, 2C). The merged pictures clearly show that the FluoTag®-X4 anti-GFP staining results in a uniform colocalization, whereas the conventional staining generates fluctuations in different compartments of the microglia. The nucleus shows a bright native GFP signal but only very faint antibody staining (figure 2A-C, star), which could be explained by worse penetration of the conventional antibodies into the nucleus or steric hindrance. In contrast, the processes of the microglia appear much brighter with the antibody staining compared to the GFP signal (figure 2A-C, arrow), most probably due to the higher amplification by secondary reagents. Scatter plots are another useful tool to visualize colocalization (ImageJ, Dunn et al., 2011). Here, the intensities of the colors are plotted against each other for each pixel (figure 1D, 2D). At high colocalization the points cluster around a straight line, as can be nicely seen in figure 1D.

FluoTag®-X4 anti-GFP-N0304-At542, GFP single domain antibody staining IHC
Conventional anti-GFP antibody staining IHC

Figure 1: Direct immunostaining with the single domain FluoTag®-X4 anti-GFP antibody. (A) Native GFP signal, (B) FluoTag®-X4 anti-GFP antibody (AB) staining, (C) merged picture and (D) scatter plot with Pearson’s correlation coefficient (PCC).

Figure 2: Indirect immunostaining with conventional anti-GFP antibody. (A) Native GFP signal, (B) Conventional antibody (AB) staining, (C) merged picture and (D) scatter plot with the Pearson’s correlation coefficient (PCC). 

Both visualizations, the merged picture and the scatter plot, suggest that the correlation of the FluoTag®-X4 anti-GFP staining with the native GFP signal is higher compared to the conventional staining. Next, the correlation was quantified with the Pearson’s correlation coefficient (PCC), which is a robust tool and not influenced by gain or offset (ImageJ, Adler et al., 2010, Bolte et al., 2006). In case of total positive correlation the PCC is 1, if there is no correlation the PCC is 0. Quantification shows an impressive correlation of the PCC=0.9732 for the FluoTag®-X4 anti-GFP-At542 and the native GFP signal (figure 1D), whereas the PCC of the conventional indirect antibody staining with the native GFP signal is only 0.7676 (figure 2D). The analysis of three pictures per group confirmed the result with high statistical significance of p< 0.001 (figure 3).

FluoTag®-X4 anti-GFP-N0304-At542, GFP single domain antibody

Figure 3: Quantification with the Pearson’s correlation coefficient (PCC) shows a significant higher correlation of the single domain FluoTag®-X4 anti-GFP antibody (AB) with the native GFP signal compared to indirect conventional immunostaining. Bar graph showing the PCC of 3 pictures per group; mean ± standard deviation given; two-tailed Student’s t-test applied.

Figure 3: Quantification with the Pearson’s correlation coefficient (PCC) shows a significant higher correlation of the single domain FluoTag®-X4 anti-GFP antibody (AB) with the native GFP signal compared to indirect conventional immunostaining. Bar graph showing the PCC of 3 pictures per group; mean ± standard deviation given; two-tailed Student’s t-test applied.

This experiment illustrates one more time that small directly conjugated single domain antibodies with monovalent binding properties show very good tissue penetration and produce staining patterns which accurately reflect the given protein expression level and distribution. They are not biased through high amplification by secondary reagents, which makes them superior for quantitative colocalization studies compared to conventional indirect immunostainings.

References

  • Adler et al., 2010: Quantifying Colocalization by Correlation: The Pearson Correlation Coefficient is Superior to the Mander’s Overlap Coefficient. PMID: 20653013
  • Bolte et al., 2006: A guided tour into subcellular colocalization analysis in light microscopy. PMID: 17210054
  • Dunn et al., 2011: A practical guide to evaluating colocalization in biological microscopy. PMID: 21209361
  • Jung et al., 2000: Analysis of Fractalkine Receptor CX3CR1 Function by Targeted Deletion and Green Fluorescent Protein Reporter Gene Insertion. PMID: 10805752
  • Prasher et al., 1992: Primary structure of the Aequorea victoria green-fluorescent protein. PMID: 1347277

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