Daniel:Notebook/HiResChrPaint/2013-7-29: Difference between revisions
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>Djacobse (Created page with "=FISH= Back to Calendar ==FISH Slide Preparation== *Followed Original protocol 1. Rinse slide...") |
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*Followed [[Daniel:Protocols/FISH#Slide Preparation|Original protocol]] | *Followed [[Daniel:Protocols/FISH#Slide Preparation|Original protocol]] | ||
1. Rinse slides in 100% ethanol; allow slides to dry completely | 1. Rinse slides in 100% ethanol; allow slides to dry completely | ||
2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media | 2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media | ||
3. Add 100 uL of cell suspension per slide roughly in the center | 3. Add 100 uL of cell suspension per slide roughly in the center | ||
4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line | 4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line | ||
5. Rinse slides briefly in 1X PBS | 5. Rinse slides briefly in 1X PBS | ||
6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS | 6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS | ||
7. Rinse briefly in 1X PBS in a coplin jar | 7. Rinse briefly in 1X PBS in a coplin jar | ||
8. Incubate for 5 minutes in 2X SSCT | 8. Incubate for 5 minutes in 2X SSCT | ||
9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide | 9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide | ||
10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C | 10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C | ||
11. Slides are typically best used within 1-2 weeks of creation | 11. Slides are typically best used within 1-2 weeks of creation | ||
Latest revision as of 18:03, 30 July 2013
FISH[edit]
FISH Slide Preparation[edit]
- Followed Original protocol
1. Rinse slides in 100% ethanol; allow slides to dry completely 2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media 3. Add 100 uL of cell suspension per slide roughly in the center 4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line 5. Rinse slides briefly in 1X PBS 6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS 7. Rinse briefly in 1X PBS in a coplin jar 8. Incubate for 5 minutes in 2X SSCT 9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide 10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C 11. Slides are typically best used within 1-2 weeks of creation