Stellaris® RNA FISH  Protocol for Adherent Cells
Grow cells on 18 mm round #1 coverglass in a 12-well cell culture plate.
Aspirate growth medium, and wash with 1 mL of 1X PBS.
Add 1 mL of fixation buffer.
Incubate at room temperature for 10 minutes.
Wash with 1 mL of 1X PBS.
Wash again with 1 mL of 1X PBS.
To permeabilize, immerse cells in 1 mL of 70% (vol./vol.)
ethanol for at least 1 hour at +2 to +8 °C.
If frozen before using, warm the reconstituted probe solution to room temperature.
Mix well by vortexing, then centrifuge briefly.
To prepare the hybridization buffer containing probe, add 1 µL of probe stock solution to 100 µL of hybridization buffer, and then vortex and centrifuge, which is enough for one coverslip.
This creates a working probe solution of 125 nM.
This solution will be used in the steps below.
Aspirate the 70% ethanol off the coverglass containing adherent cells within the 12-well plate.
Add 1 mL of Wash Buffer A (see recipe in guidelines), 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.
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.
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, 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.
Proceed to imaging.
