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 .
