Name: Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue
Uploaded: 2023-10-06T12:20:00
Description: Watch this Scientific Journal Video about Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue at JoVE.com

post-expansion biomolecule profiling of the digested and expanded tissue

Ekspansionsmikroskopi til robust 11-dobbelt udvidelse

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).

Forfatter Spotlight: Universel molekylær retention med 11 gange ekspansionsmikroskopi  

Universal molekylær retention med 11 gange ekspansionsmikroskopi

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 Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue at JoVE.com

Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue  

Post-ekspansion biomolekyleprofilering af det fordøjede og ekspanderede væv

Til at begynde med skal du tage den fordøjede og udvidede arkiverede eller friske kliniske vævsprøve og fortsætte med antistoffarvning ved at placere den i en enkelt brønd i en seks-brønds cellekulturplade. Prøven vaskes tre gange med PBS i 10 minutter hver ved stuetemperatur. Afhængigt af prøvestørrelsen tilsættes 0,5 til 2 ml af de primære antistoffer i farvningsbufferen for at dække gelen tilstrækkeligt.Inkuber natten over ved 37 grader Celsius. Derefter vaskes prøven tre gange med PBS i 10 minutter hver ved stuetemperatur. Tilsæt de tilsvarende sekundære antistoffer og inkuber i tre timer ved 37 grader Celsius i den valgte farvningsbuffer.Vask prøven igen som vist tidligere, og tilsæt 1 til 2 ml polyethylenglycol på prøven i en plade med seks brønde. Plet prøven med fluor 4 konjugeret NHS-ester i tre timer ved stuetemperatur. Ved inkubationens afslutning vaskes prøven tre gange med PBS.For at udføre lipidfarvning skal du derefter tage den udvidede vævsprøve, der vaskes tre gange i PBS, anvende et konventionelt lipofilt farvestof fortyndet 200 gange i 2 ml 0,1% Triton X-100 eller PBS i 72 til 96 timer ved stuetemperatur og vask mindst tre gange med PBS. For at udføre FISH skal du forberede hybridiseringsbufferen ved at kombinere 2 x SSC, 10% dextransulfat, 20% ethylencarbonat og 0,1% Tween 20. Anbring homogeniserede gelprøver i en hybridiseringsbuffer, der er forvarmet til 60 grader Celsius i 30 minutter.Derefter inkuberes gelprøverne med en hybridiseringsbuffer indeholdende 10 picomolære FISH-sonder mod målgenet natten over ved 45 grader Celsius. Prøverne vaskes med streng vaskebuffer to gange i 15 minutter hver ved 45 grader Celsius og to gange i 10 minutter ved 37 grader Celsius. Prøven vaskes tre gange med PBS i 10 minutter og videreformidles eller opbevares i PBS plus 0,02 % natriumazid ved 4 grader Celsius.For fuldt ud at udvide og afbilde vævet skal du flytte prøverne udvidet fire gange i PBS til en billeddannelsesplade med seks brønde i glasbund. Hvis prøven er blevet udvidet yderligere, skæres den i mindre stykker. Påfør 0,1% polylysin på glasoverfladen.Placer prøven på objektglasset, og fjern overskydende væske omkring gelen med en laboratorieklud af papir for at forhindre gelbevægelse under billeddannelsen. Udfør derefter fluorescensbilleddannelse ved hjælp af et konventionelt bredfeltmikroskop, et konfokalmikroskop eller et andet billeddannelsessystem efter eget valg. Efter billeddannelse krympes prøverne i PBS med mindst tre 10-minutters vaske, da langtidsopbevaring i deioniseret vand fører til nedbrydning af fluorescerende signal.Til sidst opbevares prøverne i PBS med 0,02 % natriumazid ved 4 grader Celsius. Konfokale billeder af fuldt udvidet musehjernevæv tillod visualisering af proteiner og celle- og mitokondriemembranstrukturer. Nukleinsyrerne i formelt og fast paraffinindlejret humant lymfeknudevæv blev også identificeret.Efter en 3,5 gange udvidelse af nyresektionen blev den revnefri ekspanderede glomerulus set. Imidlertid resulterede utilstrækkelig forankring eller homogenisering i revner, forvrængninger og tab af mærkede mål i en anden nyresektion.

Research

JoVE Journal

Biology

Post-Expansion Biomolecule Profiling of the Digested and Expanded Tissue

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 ...