Stellaris® RNA FISH Cells in Suspension Protocol. 
Centrifuge suspension cells (2 – 5 x 106 cells) in a 15 mL conical tube.
Aspirate supernatant, leaving cells in a pellet at base of tube.
Gently resuspend cells in 1 mL of 1X PBS, and centrifuge to pellet cell suspension.
Aspirate the 1X PBS, and gently resuspend cells in 1 mL of fixation buffer.
Mix well by pipetting or inverting the tube.
Incubate at room temperature for 10 minutes.
Centrifuge to pellet cell suspension.
Aspirate fixation buffer, and wash cells three times with 1 mL of 1X PBS.
Mix well by gently pipetting up and down to resuspend pellet.
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 step 13.
Invert tube with fixed and permeabilized suspension cells several times to resuspend cells.
Then place 50- 500 μL of cells (depending on concentration) in a microcentrifuge tube.
Alternatively, at this step you can use poly-L-lysine or cytospin to adhere the fixed and permeabilized suspension cells to a round #1 coverglass after which you can perform RNA FISH following the Adherent Cell Protocol.
Centrifuge to pellet cells and aspirate 70% ethanol.
Gently resuspend cells in 500 μL of Wash Buffer A (see recipe above).
Centrifuge to pellet cells and aspirate Wash Buffer A. 
Resuspend cells in 100 μL of Hybridization Buffer containing probe.
Mix well by pipetting up and down.
Incubate microcentrifuge tube in the dark at 37 °C overnight (~16 hours).
Centrifuge to pellet cells and aspirate about 50% of the Hybridization Buffer containing probe.
The pellet is very fluffy and easy to lose at this point.
Add 500 μL of Wash Buffer A.
Centrifuge to pellet cells and aspirate solution.
Be careful not to disturb the pellet.
Resuspend cells in 500 μL of Wash Buffer A. 
Incubate in the dark at 37 °C for 30 minutes.
Centrifuge to pellet cells and aspirate Wash Buffer A. 
Resuspend cells in 500 μL of DAPI nuclear stain (1X Wash Buffer A consisting of 5 ng/mL DAPI) to counterstain the nuclei.
Incubate in the dark at 37 °C for 30 minutes.
Centrifuge to pellet cells and aspirate DAPI nuclear stain.
Resuspend cells in 500 μL of Wash Buffer B.
Centrifuge to pellet cells and aspirate Wash Buffer B. 
Resuspend cells in a small drop (approximately 30 μL) of Vectashield Mounting Medium.
Place 5-10 μL of cell suspension on a clean glass microscope slide and then place an 18 x 18 mm square #1 coverglass over the cells to spread the solution.
Place a Kimwipe over the coverglass and apply gentle pressure over the surface of the coverglass, pressing it firmly onto the surface of the slide.
While applying pressure, be careful not to move the coverglass horizontally as this could result in sheared cells.
The Kimwipe will wick up excess mounting medium.
Seal the coverglass perimeter with clear nail polish, and allow to dry.
Proceed to Imaging.
