Name: Sample Digestion and Tissue Expansion of Polymerized Tissue
Uploaded: 2023-10-06T12:20:00
Description: Watch this Scientific Journal Video about Sample Digestion and Tissue Expansion of Polymerized Tissue at JoVE.com

sample digestion and tissue expansion of polymerized tissue

Expansion Microscopy for Robust 11-Fold Expansion

Biological systems&#160;exhibit structural heterogeneity, from the limbs and the organs down to the levels of proteins at the nanoscale. Therefore, a complete understanding of the operation of these systems requires visual examination across these size scales. However, the diffraction limit of light causes challenges in visualizing structures smaller than ~200-300 nm on a conventional fluorescence microscope. In addition, optical super-resolution methods1,2,3, such as stimulated emission depletion (STED), photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and structured illumination microscopy (SIM), though powerful, present their own challenges, as they require expensive hardware and reagents and often have slow acquisition times and a poor ability to image large volumes in 3D.
Expansion microscopy4 (ExM) provides an alternative means of circumventing the diffraction limit of light by covalently anchoring biomolecules into a water-swellable polymer gel and physically pulling them apart, thus rendering them resolvable on conventional optical microscopes. A multitude of ExM protocol variants have been developed since the original publication of&#160;ExM less than a decade ago, and these protocols allow the direct incorporation of proteins5,6,7, RNA8,9,10, or lipids11,12,13 into the gel network by altering the chemical anchor or expanding the sample further (thus improving the effective resolution) either in a single step14 or multiple iterative steps15,16. Until recently, no single ExM protocol could retain these three biomolecule classes with a single commercially available chemical anchor while providing a mechanically sturdy gel that could expand ~10-fold in a single expansion round.
Here, we present Magnify17, a recent addition to the ExM arsenal that uses methacrolein as the biomolecule anchor. Methacrolein forms covalent bonds with tissue like that of paraformaldehyde, ensuring that multiple classes of biomolecules can be retained within the gel network without requiring various specific or custom anchoring agents. Additionally, this technique can expand a broad spectrum of tissues up to 11-fold, including notoriously challenging samples such as formalin-fixed paraffin-embedded (FFPE) clinical samples. Previous methods for expanding such mechanically rigid samples required harsh protease digestion, rendering the antibody labeling of proteins of interest impossible after the sample had been expanded. In contrast, this technique achieves the expansion of FFPE clinical samples using a hot denaturing solution, thus preserving whole protein epitopes within the gel, which can be targeted for post-expansion imaging (Figure 1).

Author Spotlight: Universal Molecular Retention with 11-Fold Expansion Microscopy  

Universal Molecular Retention with 11-Fold Expansion Microscopy

We're trying to find easy and low cost ways for researchers to image their biological specimens at resolutions below the diffraction limit, particularly in the areas of neuroscience and clinical pathology. Many recent expansion microscopy developments involve modifying gel chemistry to expand samples further, changing the anchoring molecule to visualize different targets and utilizing heating alteration to preserve protein epitopes. With innovated magnify, a robust expansion microscopy method that allows nanoscale imaging of various biological or clinical specimens.It simplifies super resolution microscopy by eliminating specialized equipment needs while preserving intricate biological features with up to 15 nanometers resolution, hence increasing accessibility and utility in labs worldwide. Previously users had to experiment with different expansion microscopy techniques, depending on the tissue and biomolecules they wanted to image. Magnify enables high resolution imaging across various biomolecules and tissue types without requiring specific anchoring steps or specialized equipment, thus bridging a significant gap in nanoscale imaging.Magnify expands tougher clinical samples over fourfold while preserving protein epitopes enabling more accurate investigation of queuing disease. Its chemical anchor also preserves a wide range of biomolecules, making it useful for basic research in animal models Magnify simplifies nanoscale imaging of biomolecules and intact specimens without complex optics or specialized equipment. It's adaptable across various imaging modalities and expansion microscopy strategies, allowing accessible cost-effective nanoscale imaging that may accelerate drug treatments and disease studies while potentially transforming tissue atlas generation.Magnify offers endless possibilities for research. Current focus areas include expanding tissue site, investigating pathological human samples at the nanoscale and studying the smallest scale changes on brain during learning and disease.

Watch this Scientific Journal Video about Sample Digestion and Tissue Expansion of Polymerized Tissue at JoVE.com

Sample Digestion and Tissue Expansion of Polymerized Tissue  

To begin, take a slide with polymerized archived fresh tissue or paraformaldehyde fixed mouse brain tissue. Wearing eye protection, use a razor blade to separate the two glass slides of the gelling chamber. Trim the blank gel around the tissue to minimize the volume and ensure to cut asymmetrically to track the orientation of the gel after homogenization.Gently lift the tissue containing gel from the slide with a razor blade. Transfer it into a two milliliter centrifuge tube. Fill the tube to the top with homogenization buffer preheated to 80 degrees Celsius.Then incubate the sample with shaking at 80 degrees Celsius for eight to 60 hours. Pour the contents of the centrifuge tube into a single well of a six well plastic cell culture plate and remove the denaturation buffer with a transfer pipette. To completely remove the remaining SDS from the hydrogel, wash the sample in a 1%non-ionic surfactant solution.Finally, store the sample in PBS and 0.02%sodium azide at four degrees Celsius.

Research

JoVE Journal

Biology

The nanoscale imaging of biological specimens can improve the understanding of disease pathogenesis. In recent years, expansion microscopy (ExM) has been ...