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 .
