Daniel:Protocols/FISH: Difference between revisions

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''Original Protocol from Beliveau et al'': [[File:Interphase_FISH_protocol.pdf]]
''Original Protocol from Beliveau et al'': [[File:Interphase_FISH_protocol.pdf]]
1. Allow stored slides to warm to room temperature
2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar
3. Transfer slides to a coplin jar containing 2X SSCT + 50% formamide at 60C; incubate 20 minutes

Revision as of 16:41, 24 July 2013

Fluorescence in situ Hybridization (FISH)

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Buffers

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

Hybridization

Original Protocol from Beliveau et al: File:Interphase FISH protocol.pdf

1. Allow stored slides to warm to room temperature
2. Incubate slides in 2X SSCT +50% (v/v) formamide for 2.5 minutes at 92 C in a pre-warmed coplin jar
3. Transfer slides to a coplin jar containing 2X SSCT + 50% formamide at 60C; incubate 20 minutes