Stellaris® RNA FISH Sequential IF + FISH in Adherent Cells Protocol
Grow cells on 18 mm round # 1 coverglass in a 12 - well cell culture plate .
Add 1 mL of fixation buffer .
Incubate at room temperature for 10 minutes .
Wash twice with 1 mL of 1X PBS .
To permeabilize , immerse cells in 1 mL of 0.1 % Triton X - 100 in 1X PBS for 5 minutes at room temperature .
Wash with 1 mL of 1X PBS .
Add 1 mL of appropriately diluted primary antibody in 1X PBS .
Incubate at room temperature for 1 hour .
Wash with 1 mL of 1X PBS for 10 minutes , and repeat 2 more times .
Add 1 mL of appropriately diluted secondary antibody in 1X PBS .
Incubate at room temperature for 1 hour .
Wash with 1 mL of 1X PBS for 10 minutes , and repeat 2 more times .
Add 1 mL of fixation buffer .
Incubate at room temperature for 10 minutes .
Wash twice with 1 mL of 1X PBS .
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 20 and 21 .
Aspirate the 1X PBS off the coverglass containing adherent cells within the 12 - well plate .
stellarisAdd 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 ) .
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 .
