ACT-PRESTO (active clarity technique–pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.
Total time
~3–5 days per tissue sample (including polymerization, clearing, and immunolabeling)
Steps
1
Infuse and polymerize hydrogel monomers in tissue
Prepare tissue samples by infusing them with hydrogel monomer solution and initiating polymerization to create a stable gel matrix throughout the tissue. This step anchors proteins in place for subsequent clearing.
▶ 00:43
2
Clear tissue via electrophoretic lipid removal
Apply electrophoretic tissue clearing (ETC) to rapidly remove lipids and render the tissue optically transparent. This active clearing approach significantly accelerates clearing compared to passive diffusion alone.
▶ 01:42
3
Deliver antibodies via pressure-assisted immunolabeling
Perform PRESTO immunolabeling by introducing primary and secondary antibodies into cleared tissue using pressure-assisted delivery to overcome diffusion barriers in dense tissues. Enables rapid, multiplex antibody penetration and binding.
▶ 02:37
4
Validate immunolabeling and prepare for imaging
Confirm successful antibody penetration and labeling of target structures throughout the cleared tissue volume. Demonstrates readiness of samples for whole-tissue or volumetric imaging analysis.
▶ 05:24