Daniel:Protocols/Stellaris
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FISH with Stellaris Probes[edit]
Methods for using the custom probes designed using Stellaris techonology.
Storage[edit]
Probes can be stored at 4C for around 1 month. Longer storage they recommend storage at -20C. In both cases, they should be dissolved (to 12.5 uM) in 400 uL TE buffer.
Hybridization[edit]
Day 1
- Aspirate the 70% ethanol off the coverglass containing adherent cells within the 12-well plate
- Add 1 mL of Wash Buffer A (see recipe above), and incubate at room temperature for 2-5 minutes
- Assemble humidified chamber: 150 mm tissue culture plate; bottom lined evenly with a flat water-saturated paper towel and a single layer of Parafilm® placed on top of the paper towel. This chamber will help prevent evaporation of the probe solution from under the coverglass
- Within the humidified chamber, dispense 100 μL of the Hybridization Buffer containing probe onto the Parafilm
- Gently transfer the coverglass, cells side down, onto the 100 μL drop of Hybridization Buffer containing probe
- Cover the humidified chamber 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)
Day 2
- Gently transfer the coverglass, cells side up, to a fresh 12-well plate containing 1 mL of Wash Buffer A
- Incubate in the dark at 37 °C for 30 minutes
- Aspirate the Wash Buffer A, and then add 1 mL of 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
- Aspirate the DAPI staining buffer, and then add 1 mL of Wash Buffer B. Incubate at room temperature for 2-5 minutes
- Add a small drop (approximately 15 μL) of Vectashield Mounting Medium onto a microscope slide, and mount coverglass onto the slide, cells side down
- Gently wick away excess anti-fade from the perimeter of the coverglass
- Seal the coverglass perimeter with clear nail polish, and allow to dry
- If necessary, gently wipe away any dried salt off the coverglass with water
Buffers[edit]
- Hybridization Buffer
- 900 μL Stellaris RNA FISH Hybridization Buffer
- 100 μL Deionized Formamide
- Wash Buffer A
- 2 mL Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60)
- Add 7 mL Nuclease-free water
- Add 1 mL Deionized Formamide
- Mix well by vortexing gently
- Wash Buffer B
- Add 88 mL of Nuclease-free water to bottle (Biosearch Technologies Cat# SMF-WB1-20) before use.
- Mix thoroughly
- TE Buffer
- 10mM Tris-HCl
- 1 mM EDTA
- pH 8.0