Daniel:Notebook/RNAFISH/2014-8-31

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Trial Run (Started 08/29/2014)

Back to Calendar

Dye Labeling

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL solvent; vortex ≥10 seconds
    1. dye cannot be saved for later use. Use immediately!
  5. Add 2 uL dye to sample
  6. Incubate in the dark for 1 hour
  7. Add 5 uL 3M NaOAc and 40 uL nfH20 to sample
  8. Recommend column purification after incubation

Pretreatment

  1. Remove slides from 4C, let them thaw and air-dry at RT for 30–60 min
  2. Re-hydrate sections in 10 mM sodium citrate buffer for 5 min
  3. Transfer slides into a Coplin jar with prewarmed (80°C) 10 mM sodium citrate buffer in a water bath and incubate there for 5–25 min
  4. Remove the Coplin jar from the water bath and let it cool down at RT
  5. Incubate the slides in 2× SSC for 5 min
  6. Transfer slides into 50% formamide in 2× SSC and incubate for at least 1 h

Hybridization

  1. Remove slide from the formamide and wipe the liquid from the back of the slide and from the face side of the slide
    1. Avoid marked section area
  2. Prepare glass hybridization chambers as follows:
    1. Roll a small piece of mounting putty (Blu Tack) into a snake.
    2. Wrap the mounting putty around tissue section, covering 3 sides
    3. Size of the chamber depends on a section size: chamber should fit to the section size as closely as possible in order to save DNA probe
  3. Place coverslip over putty, press gently to seal the coverslip to the putty
  4. Load the probe under chamber from its open side so that probe fills in the whole space above the section
  5. Seal the chamber with rubber cement
    1. It is important that rubber cement covers the entire chamber periphery or even the whole chamber
  6. Let the rubber cement dry at RT
    1. From this step, protect slide from light
  7. Place slide on a hot-block (or water bath) with temperature 45°C for 1–3 h in order to allow infiltration of the section with the probe
  8. Place slide on a hot-block at 80°C for 5 min for the probe and section denaturation of DNA
  9. Hybridize slide for 2 or 3 days at 37°C in a waterbath or temperature controlled oven