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==Hybridization== '''Day 1''' #Aspirate the 70% ethanol off the coverglass containing adherent cells within the 12-well plate #Add 1 mL of Wash Buffer A (see recipe above), and incubate at room temperature for 2-5 minutes #Assemble humidified chamber: 150 mm tissue culture plate; bottom lined evenly with a flat water-saturated paper towel and a single layer of Parafilm® placed on top of the paper towel. This chamber will help prevent evaporation of the probe solution from under the coverglass #Within the humidified chamber, dispense 100 μL of the Hybridization Buffer containing probe onto the Parafilm #Gently transfer the coverglass, cells side down, onto the 100 μL drop of Hybridization Buffer containing probe #Cover the humidified chamber with the tissue culture lid, and seal with Parafilm #Incubate in the dark at 37 °C for at least 4 hours. (Incubation can be continued up to 16 hours) '''Day 2''' #Gently transfer the coverglass, cells side up, to a fresh 12-well plate containing 1 mL of Wash Buffer A #Incubate in the dark at 37 °C for 30 minutes #Aspirate the Wash Buffer A, and then add 1 mL of DAPI nuclear stain (Wash Buffer A consisting of 5 ng/mL DAPI) to counterstain the nuclei #Incubate in the dark at 37 °C for 30 minutes #Aspirate the DAPI staining buffer, and then add 1 mL of Wash Buffer B. Incubate at room temperature for 2-5 minutes #Add a small drop (approximately 15 μL) of Vectashield Mounting Medium onto a microscope slide, and mount coverglass onto the slide, cells side down #Gently wick away excess anti-fade from the perimeter of the coverglass #Seal the coverglass perimeter with clear nail polish, and allow to dry #If necessary, gently wipe away any dried salt off the coverglass with water ===Buffers=== *Hybridization Buffer **900 μL Stellaris RNA FISH Hybridization Buffer **100 μL Deionized Formamide *Wash Buffer A **2 mL Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60) **Add 7 mL Nuclease-free water **Add 1 mL Deionized Formamide **Mix well by vortexing gently *Wash Buffer B **Add 88 mL of Nuclease-free water to bottle (Biosearch Technologies Cat# SMF-WB1-20) before use. **Mix thoroughly *TE Buffer **10mM Tris-HCl **1 mM EDTA **pH 8.0
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