Daniel:Protocols/Stellaris: Difference between revisions

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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.   
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==
'''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===
*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
*TE Buffer
**10mM Tris-HCl
**10mM Tris-HCl
**1 mM EDTA
**1 mM EDTA
**pH 8.0
**pH 8.0

Latest revision as of 22:09, 11 January 2016

FISH with Stellaris Probes[edit]

Back to Main

Methods for using the custom probes designed using Stellaris techonology.

Adherent Cells Manual

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

  1. Aspirate the 70% ethanol off the coverglass containing adherent cells within the 12-well plate
  2. Add 1 mL of Wash Buffer A (see recipe above), and incubate at room temperature for 2-5 minutes
  3. 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
  4. Within the humidified chamber, dispense 100 μL of the Hybridization Buffer containing probe onto the Parafilm
  5. Gently transfer the coverglass, cells side down, onto the 100 μL drop of Hybridization Buffer containing probe
  6. Cover the humidified chamber with the tissue culture lid, and seal with Parafilm
  7. Incubate in the dark at 37 °C for at least 4 hours. (Incubation can be continued up to 16 hours)

Day 2

  1. Gently transfer the coverglass, cells side up, to a fresh 12-well plate containing 1 mL of Wash Buffer A
  2. Incubate in the dark at 37 °C for 30 minutes
  3. 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
  4. Incubate in the dark at 37 °C for 30 minutes
  5. Aspirate the DAPI staining buffer, and then add 1 mL of Wash Buffer B. Incubate at room temperature for 2-5 minutes
  6. Add a small drop (approximately 15 μL) of Vectashield Mounting Medium onto a microscope slide, and mount coverglass onto the slide, cells side down
  7. Gently wick away excess anti-fade from the perimeter of the coverglass
  8. Seal the coverglass perimeter with clear nail polish, and allow to dry
  9. 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