Stellaris® RNA FISH Protocol for Frozen Tissue
Frozen tissue must be sliced at a thickness of 4-10 μm using a cryostat and mounted onto a microscope slide.
Thaw the slide-mounted tissue section to room temperature.
Immerse the slide in fixation buffer for 10 minutes at room temperature.
Wash with 1 mL of 1X PBS for 2-5 minutes.
Wash again with 1 mL of 1X PBS for 2-5 minutes.
To permeabilize the tissue section, immerse the slide in 70% ethanol for at least 1 hour at room temperature.
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 1 µL of probe stock solution to 100 µL of Hybridization Buffer, and then vortex and centrifuge, which is enough for one coverslip.
This creates a working probe solution of 125 nM.
This solution will be used in the steps below.
Immerse the slide-mounted tissue section in 1X Wash Buffer 1 (see recipe on website) for 2-5 minutes.
Assemble humidified chamber: 150 mm tissue culture plate; a single water-saturated paper towel placed alongside the inner chamber edge.
Remove the slide from wash buffer, and carefully wipe away excess buffer surrounding the tissue section.
Dispense 100 µL of Hybridization Buffer containing probe onto the tissue section of the slide.
Carefully place a clean 18 mm square coverglass over the Hybridization Buffer containing probe to completely cover the tissue section, and allow for even distribution of the solution.
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.
Immerse the slide in Wash Buffer 1, and allow the submerged coverglass to slide off the tissue section.
Incubate in the dark at 37 °C for 30 minutes.
Decant Wash Buffer 1, and then add DAPI nuclear stain (1X Wash Buffer 1 consisting of 5 ng/mL DAPI) to counterstain the nuclei.
Incubate in the dark at 37 °C for 30 minutes.
Decant the DAPI staining buffer, and then immerse slide in Wash Buffer 2 for 2-5 minutes.
Remove the slide from Wash Buffer 2, and carefully wipe away excess buffer surrounding the tissue section.
Add a small drop (approximately 15 µL) of Vectashield Mounting Medium onto the tissue section, and cover with a clean 18 mm square #1 coverglass.
Gently squeeze out excess anti-fade from underneath the coverglass.
Seal the coverglass perimeter with clear nailpolish, and allow to dry.
Proceed to imaging.
