Stellaris® RNA FISH Alternative 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 .
To fix and permeabilize cells , add 1 mL of methanol - acetic acid ( MeOH - AcOH ) fixation solution .
Incubate at room temperature for 10 minutes .
Cells can be stored at + 2 to + 8 °C in MeOH - AcOH up to 48 hours before hybridization .
Do not use a coverglass containing adherent cells if the MeOH - AcOH has completely evaporated .
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 ( enough for one coverglass ) .
This creates a working probe solution of 125 nM .
This solution will be used on steps d and e .
Aspirate the MeOH - AcOH 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 2 hours .
( Incubation can be continued up to 16 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 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 using water .
Proceed to Imaging
