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 vortex and 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
