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

Ekspansjonsmikroskopi for robust 11 ganger utvidelse

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: Universell molekylær retensjon med 11 ganger ekspansjonsmikroskopi  

Universell molekylær retensjon med 11 ganger ekspansjonsmikroskopi

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  

Biomolekylprofilering etter ekspansjon av det fordøyde og utvidede vevet

For å begynne, ta den fordøyde og utvidede arkiverte eller ferske kliniske vevsprøven og fortsett med antistofffarging ved å plassere den i en enkelt brønn på en seks-brønns cellekulturplate. Vask prøven tre ganger med PBS i 10 minutter hver ved romtemperatur. Avhengig av prøvestørrelsen, tilsett 0,5 til 2 ml av de primære antistoffene i fargebufferen for å dekke gelen tilstrekkelig.Inkuber over natten ved 37 grader Celsius. Deretter vasker du prøven tre ganger med PBS i 10 minutter hver ved romtemperatur. Tilsett de tilsvarende sekundære antistoffene og inkuber i tre timer ved 37 grader Celsius i den valgte fargebufferen.Vask prøven på nytt som vist tidligere, og tilsett 1 til 2 ml polyetylenglykol på prøven i en seksbrønnsplate. Flekk prøven med Fluor 4 konjugert NHS-ester i tre timer ved romtemperatur. På slutten av inkubasjonen, vask prøven tre ganger med PBS.Deretter, for å utføre lipidfarging, ta den utvidede vevsprøven vasket tre ganger i PBS, påfør et konvensjonelt lipofilt fargestoff fortynnet 200 ganger i 2 milliliter 0,1% Triton X-100 eller PBS i 72 til 96 timer ved romtemperatur, og vask minst tre ganger med PBS. For å utføre FISH, klargjør hybridiseringsbufferen ved å kombinere 2 x SSC, 10 % dextransulfat, 20 % etylenkarbonat og 0,1 % mellom 20. Plasser homogeniserte gelprøver i en hybridiseringsbuffer forvarmet ved 60 grader Celsius i 30 minutter.Deretter inkuberer du gelprøvene med en hybridiseringsbuffer som inneholder 10 picomolære FISH-sonder mot målgenet over natten ved 45 grader Celsius. Vask prøvene med streng vaskebuffer to ganger i 15 minutter hver ved 45 grader Celsius og to ganger i 10 minutter ved 37 grader Celsius. Vask prøven tre ganger med PBS i 10 minutter og fortsett med avbildning eller lagring av prøven i PBS pluss 0,02% natriumazid ved 4 grader Celsius.For å utvide og avbilde vevet, flytt prøvene utvidet fire ganger i PBS til en glassbunn seks-brønns bildeplate. Hvis prøven er utvidet ytterligere, kutt den i mindre biter. Påfør 0,1% polylysin på glassoverflaten.Plasser prøven på lysbildet, og fjern overflødig væske rundt gelen med en papirlaboratorieklut for å forhindre gelbevegelse under avbildning. Etterpå utfører fluorescensavbildning ved hjelp av et konvensjonelt bredfeltmikroskop, et konfokalmikroskop eller et annet bildebehandlingssystem. Etter avbildning, krymp prøvene i PBS med minst tre 10-minutters vask, da langvarig lagring i avionisert vann fører til nedbrytning av det fluorescerende signalet.Til slutt lagrer du prøvene i PBS med 0,02% natriumazid ved 4 grader Celsius. Konfokale bilder av fullt utvidet mushjernevev tillot visualisering av proteiner og celle- og mitokondriemembranstrukturer. Nukleinsyrene i formelt og fast parafininnebygd humant lymfeknutevev ble også identifisert.Etter en 3,5 ganger ekspansjon av nyreseksjonen ble den sprekkfrie utvidede glomerulus sett. Imidlertid resulterte utilstrekkelig forankring eller homogenisering i sprekker, forvrengninger og tap av merkede mål i en annen nyreseksjon.

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 ...