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Updated: Feb 11, 2026
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ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
Published on: December 31, 2016
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Optimization of immunolabeling and clearing techniques for indelibly labeled memory traces
Ina P Pavlova1, Shannon C Shipley1, Marcos Lanio2
1Division of Integrative Neuroscience, Research Foundation for Mental Hygiene, Inc. (RFMH)/New York State Psychiatric Institute (NYSPI), New York, New York, 10032.
Hippocampus
|April 18, 2018
Summary
Researchers explored whole-brain clearing techniques to visualize memory traces (engrams) in mice. Modified CUBIC with Reagent-1A* enabled reproducible clearing and immunolabeling in tissue sections for memory trace visualization.
Area of Science:
- Neuroscience
- Molecular Biology
- Biotechnology
Background:
- Genetic tools enable visualization and manipulation of memory traces (engrams) in small brain regions.
- Visualizing engrams across the entire brain is crucial for understanding memory storage and coexistence in neural networks.
- Intact tissue clearing and imaging offer a promising approach for whole-brain engram visualization.
Purpose of the Study:
- To evaluate leading whole-brain clearing protocols for visualizing indelible memory traces in ArcCreERT2 mice.
- To identify an optimal method for reproducible clearing and immunolabeling of memory traces across the entire brain.
Main Methods:
- Application of established tissue clearing protocols (CLARITY, PACT, iDISCO, tert-Butanol, CUBIC, CUBIC with Reagent-1A) to ArcCreERT2 mouse brains.
- Assessment of tissue distortion, fluorescence preservation (EYFP), and immunolabeling efficiency (Arc IEG).
- Modification of CUBIC with Reagent-1A for enhanced clearing and labeling.
Main Results:
- CLARITY and PACT caused significant tissue distortion.
- iDISCO quenched EYFP fluorescence and impaired immunolabeling.
- tert-Butanol avoided harmful effects but did not allow whole-brain immunolabeling.
- CUBIC and CUBIC with Reagent-1A showed improved antibody penetration and EYFP preservation but not whole-brain visualization.
- Modified CUBIC with Reagent-1A* preserved EYFP and allowed Arc IEG immunolabeling in deep brain areas, but optimal visualization required mm-thick sections.
Conclusions:
- CUBIC with Reagent-1A* is the preferred method for reproducible clearing and immunolabeling of memory traces in mm-thick ArcCreERT2 mouse brain sections.
- Current whole-brain clearing methods have limitations in preserving fluorescence and enabling deep-tissue immunolabeling for engram visualization.
- Further optimization is needed for true whole-brain engram visualization without sectioning.
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