Stellaris® RNA FISH Protocol for Brain
Thaw the slide - mounted tissue section to room temperature .
Immerse the slide in cold 4 % E . M . grade paraformaldehyde in 1X PBS for 15 minutes .
Wash with 1X PBS for 5 minutes .
Wash twice with 1X PBS for 5 minutes .
Dip the slide in nuclease - free water .
Dip the slide in 1X TEA buffer .
Immerse the slide in 1X TEA + Acetic Anhydride for 10 minutes ( Stirring ! )
* * * Immerse the slide in 2X SSC for 3 minutes .
Immerse the slide in 70 % ethanol for 3 minutes .
Immerse the slide in 95 % ethanol for 3 minutes .
Immerse the slide in 100 % ethanol for 3 minutes .
Immerse the slide in Chloroform for 5 minutes .
Immerse the slide in 100 % ethanol for 3 minutes .
Immerse the slide in 95 % ethanol for 3 minutes .
Let air dry for 90 + minutes ( but no longer than 4 hours ) .
If frozen before using , warm the reconstituted probe solution to room temperature .
Mix well by vortexing , then centrifuge briefly .
To prepare the hybridization solution , add 4.0 μL of probe stock solution to 200 μL of hybridization buffer , and then vortex and centrifuge .
This creates a working probe solution of 250 nM .
This solution will be used on step 18 .
Assemble a humidified chamber : 150 mm tissue culture plate ; a single water - saturated paper towel placed alongside the inner chamber edge .
This chamber will help prevent evaporation of the probe solution from the tissue section .
After slide has dried for 90 + minutes , dispense 200 μL of hybridization buffer containing probe onto the tissue sections of the slide .
Carefully place a clean 24 x 60 mm rectangular coverglass over the hybridization solution to completely cover the tissue sections and allow for even distribution of the hybridization solution .
Place the slide in the humidified chamber , cover with the tissue culture lid , and seal chamber with parafilm .
Incubate in the dark at 37 °C for at least 4 hours ( incubation can be continued up to 16 hours ) .
Immerse the slide in wash buffer A , and allow the submerged coverglass to slide off the tissue section .
Gentle agitation maybe required to remove the coverglass .
Incubate in the dark at 37 °C for 30 minutes .
Decant wash buffer A , and then add DAPI nuclear stain ( wash buffer 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 immerse slide in Wash Buffer B for 3 minutes .
Immerse slide in 50 % ethanol for 3 minutes .
Immerse slide in 85 % ethanol for 3 minutes .
Immerse slide in 100 % ethanol for 3 minutes .
Let air dry for 5 - 10 minutes .
Add a drop or two ( approximately 50 - 100 μL ) of Prolong Gold Antifade Mountant onto the tissue sections .
Cover with a clean 24 x 60 mm coverglass , allowing the antifade to spread evenly across the tissue sections .
Allow Prolong Gold to cure overnight , in the dark , at room temperature .
Seal the coverglass perimeter with clear nail polish , and allow to dry in the dark .
Proceed to imaging .
