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.
3Immerse 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.
