ADAPT-3D™ Tissue Clearing Kit

The ADAPT-3D tissue imaging process is primarily used for high-resolution 3D fluorescence imaging of biological tissues in both research and clinical settings, enabling rapid, deep, and artifact-free visualization of tissue structures and cellular features across a broad range of species and tissue types.

Main Use Cases

• Whole-organ and large tissue imaging: ADAPT-3D is extensively used for imaging large, intact samples such as mouse brain, liver, lung, spleen, and skull-brain interfaces, enabling detailed 3D visualization without significant tissue shrinking or deformation.
• Immunolabeling and antigen detection: The protocol supports deep immunolabeling for detecting antigens (e.g., tight junction proteins, smooth muscle actin, S100A9, CD163, IBA1) in thick tissues, facilitating analysis of immune cell types, tissue architecture, and disease markers at high resolution.
• Retention of fluorescence reporting: ADAPT-3D preserves endogenous fluorescent reporters (like eYFP, tdTomato) and supports antibody staining, crucial for studies reliant on fluorescence for cellular and structural identification.
• Complex structure visualization: Provides 3D insights into convoluted structures such as brain vasculature, neuronal networks, and specialized channels between the skull and brain, as well as quantification of tissue features like villus height and immune populations in intestinal tissue.
• Speed and efficiency: Offers accelerated tissue clearing (hours to a few days versus weeks), broad compatibility across tissue types, and minimal distortion, making it suitable for time-sensitive preclinical and biomedical research.
• Safe, aqueous-based protocol: Avoids toxic solvents utilized in older clearing methods, reducing safety risks and simplifying lab procedures, with primary use in microscopy labs for confocal, widefield, and light-sheet imaging.

Summary Table

In summary, ADAPT-3D is a versatile imaging platform empowering high-efficiency, safe, and high-fidelity 3D imaging of biological tissues for applications in pathology, neuroscience, immunology, and translational research.

ADAPT-3D outperforms both iDISCO and CLARITY in several critical aspects of tissue clearing for 3D imaging. It is significantly faster, avoids tissue shrinkage, preserves endogenous fluorescence, and uses less toxic reagents, making it highly attractive for advanced research applications.

Speed and Workflow

• ADAPT-3D achieves complete clearing and refractive index matching (RIM) in hours, whereas iDISCO and CLARITY generally require days.
• For example, clearing an intact perfused mouse brain takes about 4 days with iDISCO+ (with notable shrinkage), but ADAPT-3D accomplished similar or better results in far less time with no shrinkage.

Tissue Integrity and Morphology

• ADAPT-3D maintains the native morphology of tissues, actively avoiding shrinkage and other deformation. iDISCO induces significant shrinkage, which can tear delicate structures.
• CLARITY, by contrast, is associated with tissue expansion and sometimes slower clearing, but retains protein and nucleic acid content via hydrogel embedding.

Fluorescence Preservation

• ADAPT-3D offers preservation of endogenous fluorescence and is compatible with antibody staining, without the need for additional chemicals to boost reporter signal.
• iDISCO+ and CLARITY can both potentially result in some loss of fluorescence, with CLARITY reportedly decreasing GFP signal intensity and iDISCO sometimes requiring optimization for certain reporters.

Toxicity and Laboratory Safety

• ADAPT-3D avoids hazardous chemicals like dichloromethane—used in iDISCO and related protocols—making it a safer and more environmentally friendly choice.
• CLARITY also uses detergents, but without the same solvent toxicity issues as iDISCO.

Performance Summary Table

ADAPT-3D provides the fastest, safest, and most morphologically accurate workflow among these options while preserving fluorescence for advanced 3D imaging.

ADAPT-3D is broadly compatible with a wide range of antibodies and fluorophores, preserving fluorescence signals and enabling deep tissue imaging for 3D applications. This platform supports both immunolabeling and the direct detection of endogenous (genetically encoded) fluorophores, such as eYFP and tdTomato, as well as conjugated antibody staining with standard fluorophores including Dylight 649 and similar Alexa Fluor, FITC, PE, and APC dyes. The protocol specifically maintains high-intensity fluorescence and is suitable for commonly used antibody targets (e.g., claudin-11, occludin, SMA, S100A9, CD163, IBA1) in both mouse and human tissues.

Compatible Antibodies

• ADAPT-3D accommodates conventional monoclonal and polyclonal antibodies, especially those used for cellular and structural markers, such as:
  • Claudin-11, occludin (tight junction proteins)
  • Smooth muscle actin (SMA), S100A9
  • CD163, IBA1 (macrophage markers)
  • Common vascular stains (e.g., Lycopersicon Esculentum lectin [Dylight 649]).

Compatible Fluorophores

• The method preserves endogenous reporter fluorophores and is compatible with typical conjugated dyes for immunofluorescence:
  • eYFP, tdTomato (genetically encoded reporters)
  • Alexa Fluor family (e.g., 488, 568, 647)
  • Dylight dyes (e.g., Dylight 649)
  • FITC, PE, APC, and their tandem derivatives.
• Recombinant antibodies directly conjugated to Vio® dyes (such as those from Miltenyi Biotec) are also validated for 3D imaging in cleared tissues, supporting a range of colors for multiplexing: FITC, PE, APC, VioGreen, PerCP-Vio 700, Vio B515, Vio G570, Vio 780, Vio R667, Vio R720, among others.

Additional Notes

• ADAPT-3D avoids harsh organic solvents, ensuring compatibility with most commercial antibody-fluorophore combinations usually employed for 3D imaging.
• Optimal results are achieved with high-quality, well-validated primary or secondary antibodies and bright, photostable fluorophores.

In summary, ADAPT-3D supports the use of a broad array of antibodies and fluorophores—including most fluorophore-conjugated antibodies validated for 3D immunofluorescence imaging—to meet the needs of multiplexed, deep-tissue visualization projects.

Validated ADAPT-3D protocols have been applied to a diverse array of tissues and species, primarily in biomedical research involving both human and mouse samples.

Validated Tissues

• Mouse brain and skull: The protocol allows for imaging without meningeal tearing and preserves tissue morphology with high sensitivity.
• Mouse spleen, lung, and liver: Including those with endogenous fluorescent hepatocytes and lymphatic vessels.
• Mouse colon wall: Effective preparation demonstrated for 1.5 mm thick sections.
• Human intestinal tissue: Efficient antibody staining and 3D imaging achieved in less than five days.
• Human ileum and macrophage subpopulations within villi: 3D reconstruction and specific immunolabeling validated.

Validated Species

• Mouse (Mus musculus): Extensive validation across multiple organs including brain, skull, spleen, lung, liver, and gastrointestinal tissues.
• Human (Homo sapiens): Protocols validated on fixed human intestinal tissues with robust imaging and immunostaining results.
• Pig (Sus scrofa): Demonstrated preparation and imaging of relatively thick pig colon wall tissue under controlled protocol timelines.

General Notes

• The ADAPT-3D protocol is broadly described as adaptable and versatile across many tissue types and model systems, indicating potential use beyond published validations.
• The protocol avoids tissue shrinkage and is non-toxic, making it suitable for fluorescence imaging of antibody-labeled and endogenous proteins through light-sheet microscopy, confocal, and related imaging techniques.

Researchers interested in 3D fluorescence imaging of fixed tissues, especially in pathology or neuroscience, have widely adopted ADAPT-3D for rapid, efficient, and morphologically-preserving clearing across animal and human samples.