Stellaris® RNA FISH FFPE (Paraffin-Embedded Tissue) Protocol
Paraffin embedded tissue must be sliced at a thickness of 4-10 μm using a microtome and mounted onto a microscope slide.
Immerse the slide-mounted tissue section in 100% xylene for 10 minutes; repeat in fresh 100% xylene for an additional 5 minutes.
Immerse the slide in 100% ethanol for 10 minutes; repeat in fresh 100% ethanol for an additional 10 minutes.
Immerse the slide in 95% ethanol for 10 minutes.
To permeabilize the tissue section, immerse the slide in 70% ethanol for at least 1 hour at room temperature.
Slides can be stored at +2 to +8 °C in 70% ethanol up to a week before hybridization.
Immerse the slide in 1X PBS for 2-5 minutes.
Decant PBS, and immerse the slide in pre-warmed (37 °C) proteinase K solution (10 μg/mL proteinase K in 1X PBS).
Incubate for 20 minutes at 37 °C.
Wash with 1X PBS for 2-5 minutes.
Wash with 1X PBS for 2-5 minutes.
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 2 μL of probe stock solution to 200 μL of Hybridization Buffer (enough for one coverslip), and then vortex and centrifuge.
This creates a working probe solution of 125 nM.
This solution will be used on steps 16 and 17.
Immerse the slide-mounted tissue section in Wash Buffer A (see recipe above) for 2-5 minutes.
Assemble 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.
Remove the slide from Wash Buffer A, and carefully wipe away excess buffer surrounding the tissue section.
Dispense 200 μL of Hybridization Buffer containing probe onto the tissue section of the slide.
(Note that 200 μL is recommended when using a 24 mm x 60 mm, rectangular, #1 coverglass.
If different sized coverglasses are used, the volume may need to be adjusted accordingly).
Carefully place a clean coverglass over the Hybridization Buffer containing probe to completely cover the tissue section, and allow for even distribution of the Hybridization Buffer.
Place the slide in the humidified chamber, cover with the tissue culture lid, and seal 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 may be 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 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 immerse slide in Wash Buffer B for 2-5 minutes.
Remove the slide from Wash Buffer B, and carefully wipe away excess buffer surrounding the tissue section.
Add a small drop (approximately 50-100 μL) of Vectashield Mounting Medium onto the tissue section, and cover with a clean #1 coverglass.
Gently squeeze out excess anti-fade from underneath the coverglass.
Seal the coverglass perimeter with clear nail polish, and allow to dry.
Proceed to Imaging.
