Daniel:Notebook/HiResChrPaint/2014-8-10

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FISH with DAPI[edit]

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Slide Preparation[edit]

  1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
  2. Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI
  3. Allow cells to adhere for 6 hours at 37 C
  4. Rinse slides briefly in 1X PBS
  5. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
  6. Rinse briefly in 1X PBS
  7. Incubate for 5 minutes in 1 mL of 2X SSCT
  8. Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
  9. Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
  10. Slides are typically best used within 1-2 weeks of creation

Hybridization[edit]

Sample Matrix

Sample Cot1-488 (below) (ug) Dystrophin-488 (pmol) Dystrophin-546 (pmol) Cot1 (ug)
A 0 8.6 (30) 0 2.5 (2.5)
B 0 0 7.9 (30) 2.5 (2.5)
C 8.5 (0.4) 0 0 0
D 8.5 (0.4) 0 7.9 (30) 0
  1. Allow stored samples to warm to room temperature
  2. Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
  3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove samples and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to center
  6. Add 22x22 #1.5 coverslip; seal with rubber cement
  7. Allow the rubber cement to air-dry for 5 minutes at room temperature
  8. Denature for 2.5 minutes at 92C; keep humid
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C