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 .
