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

Expansiemicroscopie voor robuuste 11-voudige expansie

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).

Auteur in de schijnwerpers: Universele moleculaire retentie met 11-voudige expansiemicroscopie  

Universele moleculaire retentie met 11-voudige expansiemicroscopie

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  

Monstervertering en weefselexpansie van gepolymeriseerd weefsel

Maak om te beginnen een dia met gepolymeriseerd gearchiveerd vers weefsel of paraformaldehyde gefixeerd muizenhersenweefsel. Draag oogbescherming en gebruik een scheermesje om de twee glasplaten van de geleerkamer te scheiden. Knip de blanco gel rond het weefsel om het volume te minimaliseren en zorg ervoor dat u asymmetrisch snijdt om de oriëntatie van de gel na homogenisatie te volgen.Til het weefsel met gel voorzichtig van de dia met een scheermesje. Breng het over in een centrifugebuis van twee milliliter. Vul de buis tot aan de bovenkant met homogenisatiebuffer voorverwarmd tot 80 graden Celsius.Incubeer het monster vervolgens met schudden bij 80 graden Celsius gedurende acht tot 60 uur. Giet de inhoud van de centrifugebuis in een enkele put van een plastic celkweekplaat met zes putten en verwijder de denaturatiebuffer met een transferpipet. Om het resterende SDS volledig uit de hydrogel te verwijderen, wast u het monster in een 1% niet-ionische oppervlakteactieve oplossing.Bewaar het monster ten slotte in PBS en 0,02% natriumazide bij vier graden Celsius.

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JoVE Journal

Biology

Sample Digestion and Tissue Expansion of Polymerized Tissue

Expansion Microscopy for Robust 11-Fold Expansion

Universal Molecular Retention with 11-Fold Expansion Microscopy

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

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

Gelling the Archived or Freshly Prepared Clinical Tissue Slides

Geleeren van de gearchiveerde of vers bereide klinische weefselglaasjes

Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue

Post-expansie biomolecuulprofilering van het verteerde en geëxpandeerde weefsel