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Quantitative validation of immunofluorescence and lectin staining using reduced CLARITY acrylamide formulations
D M Krolewski1, V Kumar2, B Martin2
1Molecular and Behavioral Neuroscience Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI, 48109, USA. dkrolews@med.umich.edu.
Brain Structure & Function
|December 16, 2017
Summary
Optimizing the CLARITY technique for brain tissue clearing is crucial for visualizing fluorescent-labeled molecules. Researchers found that a specific hydrogel formulation with 4% sodium dodecyl sulfate best preserves fluorescent signals.
Area of Science:
- Neuroscience
- Biotechnology
- Microscopy
Background:
- The CLARITY technique allows 3D visualization of fluorescently labeled molecules in intact, clarified brain tissue.
- Optimizing hydrogel and detergent concentrations is key for effective tissue clearing and antibody penetration.
- The impact of varying CLARITY processing parameters on fluorescent signal retention remains largely unexplored.
Purpose of the Study:
- To optimize CLARITY processing for enhanced visualization of molecular neuroanatomy and neurocircuitry.
- To determine the ideal combination of acrylamide and sodium dodecyl sulfate concentrations for maximal fluorescent signal retention.
Main Methods:
- Quantitative parvalbvalbumin immunofluorescence and lectin-based vasculature staining were performed on mouse brain slices.
- Experiments utilized varying concentrations of acrylamide monomer, bis-acrylamide, and sodium dodecyl sulfate.
- Images were acquired using confocal and CLARITY-optimized lightsheet microscopy.
Main Results:
- A hydrogel formulation of 2% acrylamide monomer with 0.0125% bis-acrylamide, cleared with 4% sodium dodecyl sulfate, demonstrated optimal signal visualization.
- This combination outperformed other tested hydrogel monomer concentrations, lipid removal times, and detergent concentrations.
- The study provides quantitative data on the effects of CLARITY parameters on fluorescent signal retention.
Conclusions:
- The optimized CLARITY protocol (2% acrylamide, 0.0125% bis-acrylamide, 4% SDS) enhances fluorescent signal visualization in clarified mouse brain tissue.
- This finding is critical for advancing molecular neuroanatomy and neurocircuitry studies.
- Further research can build upon these optimized parameters for improved neuroimaging.
