CHAPS Urea Composition for Deep Antibody Penetration in Large Tissues
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Solution Overview
Problem
Current methods for immunostaining large biological tissues face challenges with limited antibody permeability and prolonged processing times, particularly for tissues thicker than 1 mm, due to hydrogel-hybridized forms and the need for extensive pretreatment, which reduces fluorescence brightness and damages proteins, DNA, and RNA.
Innovation Solution
A composition comprising an aqueous solution of DMSO, Triton X-100, and a primary antibody, which increases antibody permeability, allowing deep penetration into tissues up to 3 mm or more, thereby facilitating rapid and high-resolution three-dimensional bioimaging without the need for extensive pretreatment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If hydrogel support is used for tissue clearing, then tissue structure is preserved, but antibody permeability is reduced and processing time is prolonged
Solution Approach 1:
The invention changes the chemical parameters of the clearing solution by using CHAPS (a zwitterionic detergent) and Urea instead of conventional hydrogel-based clearing solutions. This parameter change allows the tissue to be cleared while maintaining protein integrity and enabling antibody penetration, thus resolving the contradiction between structure preservation and antibody permeability.
2Length of stationary object
If tissue thickness is increased to 3 mm or more for whole organ imaging, then comprehensive three-dimensional observation is achieved, but antibody penetration becomes difficult
Solution Approach 1:
The invention uses CHAPS and Urea as intermediary substances that facilitate antibody penetration into thick tissues. These chemicals act as mediators by clearing the tissue in a way that opens pathways for antibody diffusion while preserving the overall tissue architecture, enabling effective immunostaining of tissues 3 mm or thicker.
3Illumination intensity
If conventional clearing methods are used, then tissue transparency is achieved, but fluorescence brightness is reduced and proteins are damaged
Solution Approach 1:
The invention converts the potential harm of chemical clearing into a benefit by using CHAPS and Urea, which clear the tissue effectively while simultaneously preserving protein integrity and fluorescence signals. The clearing process, which could damage proteins, is transformed into a method that maintains both transparency and molecular integrity.
4Manufacturing precision
If extensive pretreatment is performed to prepare tissue for immunostaining, then staining quality is improved, but processing time is significantly prolonged
Solution Approach 1:
The invention performs preliminary tissue clearing using CHAPS and Urea before immunostaining, which prepares the tissue in advance by removing lipids and increasing permeability. This preliminary action eliminates the need for extensive additional pretreatment steps, thereby reducing total processing time while maintaining high staining quality.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The composition enables clear, high-resolution three-dimensional imaging of large biological tissues, overcoming antibody permeability limitations and reducing immunostaining time, while maintaining protein integrity, and can be used for disease analysis and drug efficacy prediction.
Implementation Method 1
The composition for immunostaining cleared large biological tissues comprises an aqueous solution in which DMSO (Dimethyl Sulfoxide), Triton X-100 and a primary antibody are dissolved... increases antibody permeability in cleared biological tissues, in particular, large biological tissues having a thickness of 3 mm or more
Implementation Method 2
A technique for realizing three-dimensional image by using not only light source but also ultrasound is actively used for diagnosis... When the concentration of the hydrogel increases, the degree of binding to the protein increases, and the structure becomes hard because a tight mesh structure is created. On the other hand, when the tissue becomes hard, it becomes difficult for the lipid to escape by surfactants
Implementation Method 3
Recently, the present inventors developed a new method using SunHyun 3D image kit based on CHAPS+Urea. This method does not require any harmful or special techniques, equipments or know-hows that the conventional methods have for tissue clearing, and can easily clear the tissue by using a solution according to the order presented in the protocol
Data Source
AI summary
The present invention relates to a composition for immunostaining cleared large tissues and a method for immunostaining cleared large biological tissues using the same. The composition for immunostaining cleared large tissues increases antibody permeability in cleared biological tissues, in particular, large biological tissues having a thickness of 3 mm or more, to allow antibodies to penetrate deep into thick tissues, thereby overcoming immunostaining problems associated with limited permeation of antibodies in existing methods, reduces the time required for immunostaining large tissues, thereby enabling simple and rapid three-dimensional bioimaging with high resolution for large biological tissues, and may be beneficially used, through structure images, for identifying the causes of various diseases, developing new treatment methods, and predicting the efficacy and toxicity of drugs. Furthermore, the composition for immunostaining cleared large tissues may be used in combination with various medical devices, and in particular, may be prepared as a kit and beneficially used as an in vitro diagnostic device.