Cubic Method
Cubic Method - Related Videos
04:31
Whole-Kidney Three-Dimensional Staining with CUBIC
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Cited by 5 •
2022
The present protocol describes a tissue clearing method and whole-mount immunofluorescent staining for three-dimensional (3D) kidney imaging. This technique can offer macroscopic perspectives in kidney pathology, leading to new biological discoveries.
09:54
Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique
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Cited by 13 •
2016
Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.
09:09
Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
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Cited by 20 •
2016
We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.
08:09
CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations
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Cited by 9 •
2016
This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.
05:50
CUBIC-based Protein Expression Visualization: A Procedure for Visualizing Protein Expression in Full Thickness Mouse Skin Biopsies
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2023
This video describes the CUBIC-based visualization technique, a tissue clearing-dependent imaging technique for visual assessment of protein expression in whole-mount skin biopsies at single-cell resolution in 3D. This technically simple protocol allows the visualization of skin proteins without preparing skin sections.
FAQs
Hydrophilic reagents are central to CUBIC processing because aminoalcohols, urea, and detergents work together to remove light-scattering lipids from specimens. This reduces optical obstruction and helps make tissue more transparent while preserving molecular labels. Their combined action supports subsequent fluorescent labeling and three-dimensional imaging across intact organs.
Tissue expansion increases the physical volume of the processed specimen, while reducing refractive-index differences improves optical compatibility within it. Together with lipid removal, these changes help light travel through cleared tissue and support visualization across large volumes. In immunology studies, the result is a broader spatial view of inflammatory or immune-cell patterns than isolated thin sections can provide.
Unlike analysis based on thin tissue sections, CUBIC preserves the relationship between labeled cells and organ-wide structure during three-dimensional imaging. Researchers can examine cellular positions throughout a large, intact specimen instead of interpreting separate slices independently. This broader spatial context is important when immune responses, pathogen localization, or tissue damage vary across different regions of an organ.
Preserving molecular labels allows fluorescent signals to remain linked to the biological features being examined after clearing. Researchers can therefore interpret where labeled cells or targets occur within the three-dimensional organ context, rather than viewing signals in disconnected slices. This spatial linkage is particularly valuable when immune responses or pathogen localization differ across tissue regions.
Processing begins with hydrophilic clearing reagents that remove light-scattering lipids and modify the specimen's optical properties. The cleared tissue is then fluorescently labeled, after which light-sheet microscopy captures information throughout the intact organ. This sequence connects chemical preparation with volumetric imaging, allowing investigators to examine cellular distributions and structural responses without relying only on thin sections.
Reducing refractive-index differences complements lipid removal during clearing by improving optical compatibility within the specimen. In CUBIC, this adjustment helps light pass through cleared tissue with less scattering-related obstruction. The practical consequence is that fluorescent signals can be examined across intact organs, making organ-wide patterns more accessible than analyses restricted to thin tissue sections.
In immunology and infection research, CUBIC can map immune-cell distribution, inflammatory responses, pathogen localization, and tissue damage across large volumes. These readouts place cellular events within organ-wide anatomy, helping investigators relate immune activity to affected tissue. The approach is suited to questions that require spatial context rather than observations from isolated tissue sections.
By linking cellular position with organ-wide structure, CUBIC supports quantitative analysis of host-pathogen interactions and disease progression. Investigators can examine pathogen localization alongside immune-cell distribution, inflammation, and tissue damage within the same large-volume context. This integrated view helps reveal how these features are spatially related across an organ during infection-associated changes.