Daniel:Protocols/FISH: Difference between revisions
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==Buffer Preparation== | |||
2X SSCT: 0.3 M NaCl, 0.03 M NaCitrate, 0.1% Tween-20 | |||
==Slide Preparation== | ==Slide Preparation== | ||
''Protocol from Beliveau et al'': [[File:Slide_preparation_for_interphase_FISH.pdf]] | ''Original Protocol from Beliveau et al'': [[File:Slide_preparation_for_interphase_FISH.pdf]] | ||
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 |
Revision as of 23:41, 23 July 2013
Fluorescence in situ Hybridization (FISH)
Buffer Preparation
2X SSCT: 0.3 M NaCl, 0.03 M NaCitrate, 0.1% Tween-20
Slide Preparation
Original Protocol from Beliveau et al: File:Slide preparation for interphase FISH.pdf
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