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. 2015 May 27:5:10640.
doi: 10.1038/srep10640.

Fast immuno-labeling by electrophoretically driven infiltration for intact tissue imaging

Jun Li  1 , Daniel M Czajkowsky  2 , Xiaowei Li  2 , Zhifeng Shao  3
Affiliations

Fast immuno-labeling by electrophoretically driven infiltration for intact tissue imaging

Jun Li et al. Sci Rep. .

Abstract

Recently developed tissue clearing techniques, where the tissue is embedded within a hydrogel, have revolutionized our ability to resolve fine cellular structures in nearly intact tissues. However, the slow rate of penetration of antibodies within this hydrogel-tissue matrix has become a significantly limiting factor in many experiments, as thick tissues often require weeks to months to be adequately labeled. Increasing the pore size of this matrix has been investigated as a possible solution, but with only modest success. Here, we have systematically examined the diffusional behavior of antibodies and other typically used immuno-labels within this hydrogel-tissue matrix and, surprisingly, found that infiltration occurs at rates similar to those of diffusion in free solution. Therefore, changing the pore size of the matrix would be expected to afford only limited improvement and, instead, some means of active transport is necessary. We show that an electrophoretically-driven approach decreases the delivery time of antibodies by more than 800-fold over simple diffusion, without incurring structural damage. These results, together with the high quality of the images obtained with this method, demonstrate the advantage of this approach, thus significantly broadening the practical range of samples that can now be investigated by whole-mount tissue clearing methods.

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Figures

Figure 1
Figure 1
The experimental setup for measuring the antibody diffusion coefficients in the clarified specimen. (a) A schematic drawing of the imaging-based assay. (b) A photograph of the specimen assembly. (c) Fluorescent image of the boxed region in (b) 70 min after the addition of fluorescently labeled Fab (red). The image of the tissue is a grey-scale version of the autofluorescence at 488 nm (excitation). (d) The fluorescence intensity profile of the red signal in (c) along the yellow line.
Figure 2
Figure 2
The diffusion profiles of the immuno-labels. (a) The measured profiles and fitted curves of IgG molecules at time points of 1, 20, 40 and 60 minutes. (b) The diffusion profiles of IgG, F(ab’)2, Fab, and nanobody after 60 min.
Figure 3
Figure 3
The calculated time (in days) for immuno-labels to completely infiltrate a typical 3 mm-thick hydrogel embedded tissue, based on our measured diffusion coefficients.
Figure 4
Figure 4
Significantly faster infiltration of IgG driven by an external electric field. (a) Fluorescent images of IgG within the clarified tissue at the indicated time points with (Electro) or without (Diff) the application of an external voltage of 25 V across the tissue-matrix. (b) The intensity profile of fluorescently labeled IgG molecules delivered by passive diffusion or the external electrical field.
Figure 5
Figure 5
Effective labeling and lack of measurable tissue damage following electrophoretically driven immuno-staining of the mouse brain section. (a) Fluorescence image of the immune-labeled Thy1-YFP mouse brain section (green: YFP, red: anti-YFP). (b) The endogenous fluorescence (i.e. from YFP) of the boxed region in (a). (c) Fluorescent image of the anti-YFP label within the boxed region in (a).
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