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

Microscopia de expansão para expansão robusta de 11 vezes

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).

Autor em destaque: Retenção molecular universal com microscopia de expansão de 11 vezes  

Retenção molecular universal com microscopia de expansão de 11 vezes

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  

Digestão de Amostras e Expansão Tecidual de Tecido Polimerizado

Para começar, pegue uma lâmina com tecido fresco polimerizado arquivado ou tecido cerebral de camundongo fixado com paraformaldeído. Usando proteção ocular, use uma lâmina de barbear para separar as duas lâminas de vidro da câmara gelificante. Corte o gel em branco ao redor do tecido para minimizar o volume e certifique-se de cortar assimetricamente para rastrear a orientação do gel após a homogeneização.Levante suavemente o tecido que contém gel da lâmina com uma lâmina de barbear. Transfira para um tubo centrífugo de dois mililitros. Encha o tubo até ao topo com tampão de homogeneização pré-aquecido a 80 graus Celsius.Em seguida, incubar a amostra com agitação a 80 graus Celsius por oito a 60 horas. Despeje o conteúdo do tubo de centrífuga em um único poço de uma placa de cultura de células plásticas de seis poços e remova o tampão de desnaturação com uma pipeta de transferência. Para remover completamente o SDS restante do hidrogel, lave a amostra em uma solução de tensoativo não iônico a 1%.Finalmente, armazenar a amostra em PBS e azida de sódio a 0,02% a quatro graus 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 ...