TITLE: No Title
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[DESCRIPTION]
<p>The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent <em>in situ</em> hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.</p>

[BEFORE_START]
<p>*Sodium Acrylate, purity note: check for yellow color upon resuspension: that indicates poor quality; solution should be clear (see <a href="http://expansionmicroscopy.org" target="_blank" rel="noopener noreferrer">http://expansionmicroscopy.org</a>)</p>
<p> </p>
<p><strong>Stock solutions: </strong></p>
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<p><em>MOPS Buffer</em>: 20 mM MOPS pH 7.7</p>
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<p>For conveniently adjusting the final volume, prepare the MOPS buffer at 10 times the final concentration (i.e. 200mM MOPS pH 7.7). When diluting LabelX in the MOPS buffer add nuclease-free water to bring the final concentration of the buffer to 20 mM MOPS pH 7.7.</p>
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<p><em>Hybridization Buffer for smFISH</em>: 10% (w/v) dextran sulfate, 10% (v/v) formamide, 2X SSC</p>
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<p>Prepare the hybridization buffer without formamide and store it at 4°C. Add formamide right before performing FISH.</p>
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<p><em>Hybridization Buffer for HCR-FISH</em>: 10% (w/v) dextran sulfate, 20% (v/v) formamide, 2X SSC</p>
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<p>Prepare the hybridization buffer without formamide and store it at 4°C. Add formamide right before FISH.</p>
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<p><em>Wash Buffer for smFISH (WA-10)</em>: 10% (v/v) formamide, 2X SSC</p>
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<p>Can be stored at room temperature for up to a week.</p>
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<p><em>Wash Buffer for HCR-FISH (WA-20)</em>: 20% (v/v) formamide, 2X SSC</p>
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<li>Can be stored at room temperature for up to a week.</li>
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<p><em>5x SSCT</em>: 5x SSC, 0.1% Tween-20</p>
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<p>Can be stored at room temperature.</p>
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<p><em>0.05x SSCT</em>: 0.05x SSC, 0.1% Tween-20</p>
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<p>Can be stored at room temperature.</p>
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<p><em>Amplification buffer</em>: 5x SSC, 10% (w/v) dextran sulfate, 0.1% Tween-20</p>
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<p>Store at 4°C.</p>
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<p><em>Digestion Buffer</em>: 50 mM Tris pH 8.0, 1 mM EDTA, 0.5% Triton X-100, 1M NaCl</p>
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<p>Can be stored at room temperature.</p>
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<p>Add Proteinase K (NEB, 1:100, final concentration 8 units/mL) to digestion buffer before use.</p>
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[GUIDELINES]
<p><strong>Preparation of LabelX:</strong></p>
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<p>Label-IT amine nucleic acid labeling reagent purchased from Mirus Bio.: <a href="https://www.mirusbio.com/products/labeling/label-it-nucleic-acid-modifying-reagent" target="_blank" rel="noopener noreferrer">https://www.mirusbio.com/products/labeling/label-it-nucleic-acid-modifying-reagent</a></p>
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<p>Resuspend Label-IT amine at 1 mg/mL using the provided resuspension buffer. Add the resuspension buffer and vortex to mix.</p>
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<p>Resuspend acryloyl X SE (AcX) (<a href="https://www.lifetechnologies.com/order/catalog/product/A20770" target="_blank" rel="noopener noreferrer">https://www.lifetechnologies.com/order/catalog/product/A20770</a>) in 500 µL anhydrous DMSO (10 mg/mL stock solution). Aliquot in 20 µL and store desiccated at -20°C.</p>
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<p>React the Label-IT to AcX at equal mass ratio. For example, add 1 µL of acryloyl X to 10 µL of Label-IT (both are resuspended in DMSO so the reaction is carried out in DMSO). React for &gt;6 hrs at room temperature on benchtop shaker. Store the LabelX at -20°C with desiccant for future use.</p>
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[STEPS]
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