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[DESCRIPTION]
A set of Stellaris FISH Probes comprises up to 48 singly labeled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris FISH Probes bound to target RNA produce fluorescent signals that permit detection of single RNA molecules as diffraction-limited spots by conventional fluorescence microscopy. See the Biosearch website for additional details.

[GUIDELINES]
<strong>General Protocol &amp; Storage <br /><br />Product Description </strong><br /><br />A set of Stellaris RNA FISH Probes is comprised of up to 48 singly labeled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris RNA FISH Probes bound to target RNA produce fluorescent signals that permit detection of single RNA molecules as diffraction-limited spots by conventional fluorescence microscopy. <br /><br /><strong>STORAGE GUIDELINES</strong><br /><br />Stellaris RNA FISH Probes <br /><ul style='list-style-type: circle;'><li>Stellaris RNA FISH Probes are shipped dry and can be stored at +2 to +8 &deg;C in this state. Dissolved probe mix should be subjected to a minimum number of freeze-thaw cycles. For daily and short-term use of dissolved probe mix, storage at +2 to +8 &deg;C in the dark for up to a month is recommended. For storage lasting longer than a month, we recommend aliquoting and freezing probes in the dark at -15 to -30 &deg;C.&nbsp;</li></ul><br />Stellaris RNA FISH Hybridization Buffer <br /><ul style='list-style-type: circle;'><li>Stellaris RNA FISH Hybridization Buffer should be stored at +2 to +8 &deg;C for short-term and long-term use.&nbsp;</li></ul><br />Stellaris RNA FISH Wash Buffer A and Wash Buffer B <br /><ul style='list-style-type: circle;'><li>Stellaris RNA FISH Wash Buffers A and B should be stored at room temperature for short-term and long-term use.&nbsp;</li></ul><br /><strong>REAGENTS and EQUIPMENT<br /></strong><br /><ul style='list-style-type: square;'><li><em>Reagents and Consumables:</em>&nbsp;</li></ul>a) 18 mm round #1 coverglass <br />b) 12-well culture plates <br />c) TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) <br />d) 37% Formaldehyde Solution <br />e) 10X Phosphate Buffered Saline (PBS), RNase-free <br />f) Nuclease-free water <br />g) Deionized Formamide <br />h) Ethanol for molecular biology <br />i) Stellaris RNA FISH Hybridization Buffer (Biosearch Technologies Cat# SMF-HB1-10) <br />j) Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60) <br />k) Stellaris RNA FISH Wash Buffer B (Biosearch Technologies Cat# SMF-WB1-20) <br />l) RNase-free consumables such as pipette tips <br />m) 4&rsquo;,6-diamidino-2-phenylindole (DAPI) <br />n) Vectashield&reg; Mounting Medium (Vector Laboratories Cat #H-1000) <br />o) Humidified chamber (or equivalent): 150 mm tissue culture plate; bottom lined evenly with a flat water-saturated paper towel and a single layer of Parafilm&reg; placed on top of the paper towel <br />p) 37 &deg;C laboratory oven <br /><br /><ul style='list-style-type: square;'><li><em>Microscope</em>:&nbsp;</li></ul>a) Wide-field fluorescence microscope (e.g., Nikon Eclipse Ti or equivalent). We provide limited support for confocal applications. <br />b) A high numerical aperture (&gt;1.3) and 60-100x oil-immersion objective. <br />c) Strong light source, such as a mercury or metal-halide lamp (Xenon or LED are typically not bright enough). <br />d) Filter sets appropriate for the fluorophores. <br />e) Standard cooled CCD camera, ideally optimized for low-light level imaging rather than speed (13 &mu;m pixel size or less is ideal). <br /><br /><strong>PREPARATION of REAGENTS<br /><br /></strong> NOTE: When performing Stellaris RNA FISH, it is imperative to limit RNA degradation. Please ensure that all consumables and reagents are RNase-free. Recipes below are for set volumes. Please adjust accordingly. <br /><br /><ul style='list-style-type: square;'><li><em>Reconstituting the dried probe stock:</em></li></ul><strong>ShipReady Probe Set (1 nmol):</strong> <br />A ShipReady probe set can provide up to 80 hybridizations. Re-dissolve the dried oligonucleotide probe blend in 80 &mu;L of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to create a probe stock of 12.5 &mu;M. Mix well by pipetting up and down, and then vortex and centrifuge briefly. <br /><br /><strong>DesignReady or Custom Probe Set (5 nmol):<br /></strong> A DesignReady or custom probe set can provide up to 400 hybridizations. Re-dissolve the dried oligonucleotide probe blend in 400 &mu;L of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to create a probe stock of 12.5 &mu;M. Mix well by pipetting up and down, and then vortex and centrifuge briefly. <br /><br /><ul style='list-style-type: square;'><li><em>Fixation buffer:&nbsp;</em></li></ul>Final composition is 3.7% (vol./vol.) formaldehyde in 1X PBS <br /><strong>For a final volume of 10 mL, mix:</strong> <br />1 mL 37% Formaldehyde solution <br />1 mL 10X Phosphate Buffered Saline (PBS), RNase-free <br />8 mL Nuclease-free water <br /><br /><ul style='list-style-type: square;'><li><em>Hybridization Buffer:</em>&nbsp;</li></ul>Final composition is 10% (vol./vol.) formamide in Hybridization Buffer <br /><br /><strong>Hybridization Buffer should be mixed fresh for each experiment:</strong> <br />Due to viscosity of the solution, we recommend accounting for a 10% final volume excess in order to have enough Hybridization Buffer for all of your samples. <br /><br /><strong>For a final volume of 1 mL, mix: </strong><br />900 &micro;L Stellaris RNA FISH Hybridization Buffer (Biosearch Technologies Cat# SMF-HB1-10) <br />100 &micro;L Deionized Formamide <br /><br /><strong>Note: Do not freeze Hybridization Buffer.</strong> <br /><br /><strong>WARNING!</strong> Formamide is a teratogen that is easily absorbed through the skin and should be used in a chemical fume hood.<br /><br /> <strong>WARNING!</strong> Be sure to let the formamide warm to room temperature before opening the bottle. <br /><br /><ul style='list-style-type: square;'><li><em>Wash Buffer A (10 mL):</em></li></ul><strong>Final composition is 10% (vol./vol.) formamide in Wash Buffer A</strong> <br />Mix and dilute Wash Buffer A fresh for each experiment:<br /> For a final volume of 10 mL, mix: <br />2 mL Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60) <br />Add 7 mL Nuclease-free water <br />Add 1mL Deionized Formamide <br />Mix well by vortexing gently. <br /><br /><ul style='list-style-type: square;'><li><em>Wash Buffer B:</em></li></ul><strong>Add Nuclease-free water to Wash Buffer B bottle upon first use.</strong> <br />Add 88 mL of Nuclease-free water to bottle (Biosearch Technologies Cat# SMF-WB1-20) before use. Mix thoroughly. <br /><br /><ul style='list-style-type: square;'><li><em>Nuclear Stain for use after hybridization:</em></li></ul>4',6-diamidino-2-phenylindole (DAPI) dissolved in Wash Buffer A (see above) at 5 ng/mL. <br /><br /><ul style='list-style-type: square;'><li><em>Mounting media:</em></li></ul>Vectashield Mounting Medium from Vector Laboratories (#H-1000). <br /><br />Note: For best results, samples mounted with Vectashield Mounting Medium should be imaged the same day.<br /><br /><strong>REFERENCES</strong><br /> 1. Raj, A., van den Bogaard, P., Rifkin S.A., van Oudenaarden, A., and Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods. 2008; 5: 877-9 <br /><br />2. Femino, A.M., Fay, F.S., Fogarty, K., and Singer, R.H. Visualization of single RNA transcripts in situ. Science 1998; 280: 585&ndash;90.

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