Daniel:Notebook/HiResChrPaint/2014-8-10
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FISH with DAPI[edit]
Slide Preparation[edit]
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in RPMI
- Add 20 uL of cell suspension per dish to the cener; add 80 uL RPMI
- Allow cells to adhere for 6 hours at 37 C
- Rinse slides briefly in 1X PBS
- Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Rinse briefly in 1X PBS
- Incubate for 5 minutes in 1 mL of 2X SSCT
- Incubate for 5 minutes in 1 mL of 2X SSCT + 50% (v/v) formamide
- Remove media and add 1 mL of 2X SSCT + 50% (v/v) formamide for storage at 4C; wrap with parafilm
- 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 |
- Allow stored samples to warm to room temperature
- Incubate in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in pre-warmed media
- Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
- Remove samples and allow to cool to room temperature
- Add 25 uL hybridization cocktail to center
- Add 22x22 #1.5 coverslip; seal with rubber cement
- Allow the rubber cement to air-dry for 5 minutes at room temperature
- Denature for 2.5 minutes at 92C; keep humid
- Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C