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[DESCRIPTION]
<p>This protocol is specifically designed for high throughput applications of Stellaris in 96 well glass bottom plates.</p>

[BEFORE_START]
<p align="LEFT">Reagents and Equipment</p>
<p align="LEFT">Reagents and Consumables:</p>
<p align="LEFT">a) TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)</p>
<p align="LEFT">b) Methanol</p>
<p align="LEFT">c) Glacial Acetic Acid</p>
<p align="LEFT">d) 37% Formaldehyde Solution</p>
<p align="LEFT">e) Ethanol for molecular biology</p>
<p align="LEFT">f) 10X Phosphate Buffered Saline (PBS), RNase-free</p>
<p align="LEFT">g) Nuclease-free water</p>
<p align="LEFT">h) Deionized Formamide</p>
<p align="LEFT">i) Stellaris RNA FISH Hybridization Buffer (LGC Biosearch Technologies Cat# SMF-HB1-10)</p>
<p align="LEFT">j) Stellaris RNA FISH Wash Buffer A (LGC Biosearch Technologies Cat# SMF-WA1-60)</p>
<p align="LEFT">k) Stellaris RNA FISH Wash Buffer B (LGC Biosearch Technologies Cat# SMF-WB1-20)</p>
<p align="LEFT">l) 4’,6-diamidino-2-phenylindole (DAPI)</p>
<p align="LEFT">m) Vectashield® Mounting Medium (Vector Laboratories Cat #H-1000)</p>
<p align="LEFT">n) 96-well glass bottom cell culture plates with #0 or #1 coverglass*</p>
<p align="LEFT">o) Mineral Oil</p>
<p align="LEFT">p) RNase free consumables such as pipette tips</p>
<p align="LEFT">q) 37 °C laboratory oven</p>
<p align="LEFT">*Cell culture plate must be resistant to the fixation, wash buffers, and microscope objective immersion oil used in this protocol</p>
<p align="LEFT">Microscope:</p>
<p align="LEFT">a) Wide-field fluorescence microscope (e.g., Nikon Eclipse Ti or equivalent). We provide limited support for confocal applications</p>
<p align="LEFT">b) A high numerical aperture (&gt;1.3) and 60-100x oil-immersion objective</p>
<p align="LEFT">c) Strong light source, such as a mercury or metal-halide lamp (newer LED-based light sources may also be sufficient)</p>
<p align="LEFT">d) Filter sets appropriate for the fluorophores</p>
<p align="LEFT">e) Standard cooled CCD camera, ideally optimized for low-light level imaging rather than speed (13 μm pixel size or less is ideal)</p>
<p align="LEFT">Preparation of Reagents</p>
<p align="LEFT">NOTE: When performing Stellaris RNA FISH, it is imperative to limit RNA degradation. Please ensure that all consumables and</p>
<p align="LEFT">reagents are RNase-free. Recipes below are for set volumes. Please adjust accordingly.</p>
<p align="LEFT">Reconstituting the dried probe stock:</p>
<p align="LEFT">ShipReady Probe Set (1 nmol): A ShipReady probe set can provide up to 50 hybridizations. Re-dissolve the dried oligonucleotide</p>
<p align="LEFT">probe blend in 80 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to create a probe stock of 12.5 μM. Mix well by pipetting</p>
<p align="LEFT">up and down, and then vortex and centrifuge briefly.</p>
<p align="LEFT">DesignReady or Custom Probe Set (5 nmol): A DesignReady or custom probe set can provide up to 250 hybridizations.</p>
<p align="LEFT">Re-dissolve the dried oligonucleotide probe blend in 400 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to create a probe</p>
<p align="LEFT">stock of 12.5 μM. Mix well by pipetting up and down, and then vortex and centrifuge briefly.</p>
<p align="LEFT">Standard Fixation Solution:</p>
<p align="LEFT">Final composition is 3.7% (vol./vol.) formaldehyde in 1X PBS</p>
<p align="LEFT">For a final volume of 10 mL, mix:</p>
<p align="LEFT">1 mL 37% Formaldehyde solution</p>
<p align="LEFT">1 mL 10X Phosphate Buffered Saline (PBS), RNase-free</p>
<p align="LEFT">8 mL Nuclease-free water</p>
<p align="LEFT">WARNING! Formaldehyde is a known carcinogen and should be used in a chemical fume hood. Please refer to the appropriate</p>
<p align="LEFT">SDS (Safety Data Sheet) prior to use.</p>
<p align="LEFT">Alternative Fixation Solution:</p>
<p align="LEFT">Final composition is 3:1 Methanol-Glacial Acetic Acid</p>
<p align="LEFT">For a final volume of 10 mL, mix:</p>
<p align="LEFT">7.5 mL Methanol</p>
<p align="LEFT">2.5 mL Glacial Acetic Acid</p>
<p align="LEFT">Hybridization Buffer:</p>
<p align="LEFT">Final composition is 10% (vol./vol.) formamide in Hybridization Buffer</p>
<p align="LEFT">Hybridization Buffer should be mixed fresh for each experiment:</p>
<p align="LEFT">Due to viscosity of the solution, we recommend accounting for a 10% final volume excess in order to have enough Hybridization</p>
<p align="LEFT">Buffer for all of your samples.</p>
<p align="LEFT">For a final volume of 1 mL, mix:</p>
<p align="LEFT">900 μL Stellaris RNA FISH Hybridization Buffer (LGC Biosearch Technologies Cat# SMF-HB1-10)</p>
<p align="LEFT">100 μL Deionized Formamide</p>
<p align="LEFT">NOTE: Do not freeze Hybridization Buffer.</p>
<p align="LEFT">WARNING! Formamide is a teratogen that is easily absorbed through the skin and should be used in a chemical fume hood.</p>
<p align="LEFT">Please consult the appropriate SDS (Safety Data Sheet) prior to use</p>
<p align="LEFT">WARNING! Be sure to let the formamide warm to room temperature before opening the bottle.</p>
<p align="LEFT">WARNING! Formaldehyde is a known human carcinogen and should be use in a chemical fume hood. Please consult the</p>
<p align="LEFT">appropriate SDS (Safety Data Sheet) prior to use</p>
<p align="LEFT">Wash Buffer A (10 mL):</p>
<p align="LEFT">Final composition is 10% (vol./vol.) formamide in 1X Wash Buffer A</p>
<p align="LEFT">Mix and dilute Wash Buffer A fresh for each experiment:</p>
<p align="LEFT">For a final volume of 10 mL, mix:</p>
<p align="LEFT">2 mL Stellaris RNA FISH Wash Buffer A (LGC Biosearch Technologies Cat# SMF-WA1-60)</p>
<p align="LEFT">Add 7 mL Nuclease-free water</p>
<p align="LEFT">Add 1 mL Deionized Formamide</p>
<p align="LEFT">Mix well by vortexing gently</p>
<p align="LEFT">Wash Buffer B:</p>
<p align="LEFT">Add Nuclease-free water to Wash Buffer B bottle upon first use.</p>
<p align="LEFT">Add 88 mL of Nuclease-free water to bottle (LGC Biosearch Technologies Cat# SMF-WB1-20) before use. Mix thoroughly.</p>
<p align="LEFT">Nuclear Stain for use after hybridization:</p>
<p align="LEFT">4',6-diamidino-2-phenylindole (DAPI) dissolved in Wash Buffer A (see above) at 5 ng/mL. This solution is to be used in Step J below.</p>
<p align="LEFT">Mounting media:</p>
<p align="LEFT">Vectashield Mounting Medium from Vector Laboratories (#H-1000).</p>
<p>NOTE: For best results, samples mounted with Vectashield Mounting Medium should be imaged the same day.</p>

[GUIDELINES]
<p align="LEFT">Storage Guidelines</p>
<p align="LEFT">Stellaris RNA FISH Probes</p>
<p align="LEFT">Stellaris RNA FISH Probes are shipped dry and can be stored at +2 to +8 °C in this state. Dissolved probe mix should be subjected</p>
<p align="LEFT">to a minimum number of freeze-thaw cycles. For daily and short-term use of dissolved probe mix, storage at +2 to +8 °C in the</p>
<p align="LEFT">dark for up to a month is recommended. For storage lasting longer than a month, we recommend aliquoting and freezing probes</p>
<p align="LEFT">in the dark at -15 to -30 °C.</p>
<p align="LEFT">Stellaris RNA FISH Hybridization Buffer</p>
<p align="LEFT">Stellaris RNA FISH Hybridization Buffer should be stored at +2 to +8 °C for short-term and long-term use.</p>
<p align="LEFT">Stellaris RNA FISH Wash Buffer A and Wash Buffer B</p>
<p>Stellaris RNA FISH Wash Buffers A and B should be stored at room temperature for short-term and long-term use.</p>

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