Stellaris® RNA FISH 96 Well Glass Bottom Plate Protocol
Grow cells in a 96-well glass bottom cell culture plate.
Decant growth medium, and wash with 200 μL of 1X PBS.
To fix cells, add 200 μL of 3.7% Formaldehyde fixation solution.
Incubate at room temperature for 10 minutes.
Wash with 200 μL of 1X PBS.
Wash with 200 μL of 1X PBS.
The Alternative Fixation steps are an alternative to the standard fixation and are not meant to be sequential.
Grow cells in a 96-well glass bottom cell culture plate. 
Decant growth media, and wash with 200 μL of 1X PBS To fix and permeabilize cells, add 200 μL 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 well if the MeOH-AcOH has completely evaporated.
If frozen before using, warm the reconstituted probe stock to room temperature.
Mix well by vortexing, then centrifuge briefly.
To prepare the Hybridization Buffer containing probe, add 1.5 μL of probe stock solution to 75 μL of Hybridization Buffer, and then vortexand centrifuge (enough for one well).
This creates a working probe solution of 250 nM.
This solution will be used on step 17.
Decant MeOH-AcOH or 70% ethanol from wells containing adherent cells.
Add 200 μL of Wash Buffer A (see recipe above), and incubate at room temperature for 2-5 minutes.
Decant Wash Buffer A Add 75 μL of Hybridization Buffer containing Probe into each well.
Incubate in the dark at 37 °C for 4 to 16 hoursa).
Incubation is recommended for 16 hours using the Standard Fixation Methodb).
Incubation is recommended for 2 hours using the Alternative Fixation Method.
Aspirate the Hybridization Buffer containing Probe, and add 200 μL of Wash Buffer A.
Incubate in the dark at 37 °C for 30 minutes.
Decant Wash Buffer A, and then add 200 μL 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.
Decant DAPI staining buffer, and then add 200 μL of Wash Buffer B.
Incubate at room temperature for 2-5 minutes.
Add 30 μL of VectaShield Mounting Medium to the well and top with 30 μL of Mineral Oil Proceed to Imaging
