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.
Highlights
We have used hydrogel-embedded mouse brain sections prepared with CLARITY as a standard specimen and systematically examined the diffusional behavior of immuno-labels of different sizes
We expect this simple protocol will significantly broaden the range of specimens that can be practically investigated with intact tissue clearing techniques
To ensure that the measurements were obtained from samples similar to those in typical experiments, we investigated 500 μ m-thick sections of clarified adult mouse brain prepared following the standard CLARITY protocol (Fig. S1)[12]
Summary
We have used hydrogel-embedded mouse brain sections prepared with CLARITY as a standard specimen and systematically examined the diffusional behavior of immuno-labels of different sizes. To monitor the diffusional behavior of various molecules in a cross-linked hydrogel-tissue matrix, we developed an imaging-based strategy in which the one-dimensional diffusional profile of fluorescently labeled molecules from the edge of the specimen is determined with confocal microscopy (Fig. 1a).
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