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[DESCRIPTION]
Stellaris® RNA FISH protocol for fresh frozen mouse brain

[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) 20% Electron Microscopy Grade Formaldehyde</p>
<p align="LEFT">c) 10X Phosphate Buffered Saline (PBS), RNase-free</p>
<p align="LEFT">d) Nuclease-free water</p>
<p align="LEFT">e) Ethanol for molecular biology</p>
<p align="LEFT">f) Triethanolamine</p>
<p align="LEFT">g) Acetic Anhydride</p>
<p align="LEFT">h) Chloroform</p>
<p align="LEFT">i) Deionized Formamide</p>
<p align="LEFT">j) Stellaris RNA FISH Hybridization Buffer (LGC Biosearch Technologies Cat# SMF-HB1-10)</p>
<p align="LEFT">k) Stellaris RNA FISH Wash Buffer A (LGC Biosearch Technologies Cat# SMF-WA1-60)</p>
<p align="LEFT">l) Stellaris RNA FISH Wash Buffer B (LGC Biosearch Technologies Cat# SMF-WB1-20)</p>
<p align="LEFT">m) 4’,6-diamidino-2-phenylindole (DAPI)</p>
<p align="LEFT">n) Prolong® Gold Antifade Mountant (ThermoFisher Scientific Cat #P36930)</p>
<p align="LEFT">o) 24 x 60 mm rectangular coverglass</p>
<p align="LEFT">p) RNase free consumables such as pipette tips</p>
<p align="LEFT">q) Humidified chamber (or equivalent): 150 mm tissue culture plate; a single water-saturated paper towel placed alongside the</p>
<p align="LEFT">inner chamber edge</p>
<p align="LEFT">r) 37 °C laboratory oven</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 80 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 400 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">Fixation Buffer:</p>
<p align="LEFT">Final composition is 3.7% (vol./vol.) formaldehyde in 1X PBS</p>
<p align="LEFT">For a final volume of 50 mL, mix:</p>
<p align="LEFT">1 mL 20% Formaldehyde solution</p>
<p align="LEFT">5 mL 10X Phosphate Buffered Saline (PBS), RNase-free</p>
<p align="LEFT">35 mL Nuclease-free water</p>
<p align="LEFT">TEA Buffer (10X):</p>
<p align="LEFT">NOTE: This buffer is light sensitive. It is suggested to wrap reagent bottle in foil.</p>
<p align="LEFT">For a final volume of 100 mL, mix:</p>
<p align="LEFT">13.3 mL Triethanolamine</p>
<p align="LEFT">60 mL Nuclease-free water H2O</p>
<p align="LEFT">Then bring to pH 8.0 using HCL (~5-10 mL of HCL)</p>
<p align="LEFT">Once at pH 8.0, add water to 100 mL final volume</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>NOTE: Do not freeze Hybridization Buffer.</p>
<p align="LEFT">Wash Buffer A (50 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 50 mL, mix:</p>
<p align="LEFT">10 mL Stellaris RNA FISH 5X Wash Buffer A (LGC Biosearch Technologies Cat# SMF-WA1-60)</p>
<p align="LEFT">Add 5 mL Deionized Formamide</p>
<p align="LEFT">Add 35 mL Nuclease-free water</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) prepared in Wash Buffer A (see above) at 5 ng/mL. This solution is to be used in Step J</p>
<p align="LEFT">below.</p>
<p align="LEFT">Mounting media:</p>
<p align="LEFT">Prolong Gold Antifade Mountant (ThermoFisher Scientific Cat #P36930).</p>
<p align="LEFT">NOTE: Samples mounted with Prolong Gold should be allowed to cure overnight at room temperature and then imaged</p>
<p>the following day.</p>
<p> </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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