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 .
