Stellaris® RNA FISH Simultaneous IF + FISH in Adherent Cells Protocol
NOTE: This protocol has been adapted for a 12-well plate system.
To adapt this protocol for your preferred system, volumesshould be adjusted accordingly.
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 twice with 1 mL of 1X PBS.
To permeabilize cells, resuspend cells in 1 mL of 70% ethanol for at least 1 hour at +2 to +8 °C.
Cells can be stored at +2 to +8 °C in 70% ethanol up to a week before hybridization.
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  12 and 13.
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 plus appropriately diluted primary antibody, onto the Parafilm.
Gently transfer the coverglass, cells side down, onto the 100 μL drop of Hybridization Buffer containing probe and primary antibody.
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 plus appropriately diluted secondary antibody.
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) plus appropriately diluted secondary antibody.
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
