Daniel:Notebook/HiResChrPaint/2014-1-27
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FISH Slide Prep
I've got cells ready to split so I'm making some dishes for FISH.
FISH Slide Preparation
Original Protocol from Beliveau et al: File:Slide preparation for interphase FISH.pdf
- Prepared 4 dishes PGP1F P9
1. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media 2. Add 100 uL of cell suspension per dish roughly in the center 3. Allow cells to adhere for 3 hours at the 37 C 4. Rinse 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 2X SSCT 8. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide 9. Add fresh 2X SSCT + 50% (v/v) formamide for storage at 4C
Pictures
I took snapshots of all four dishes after fixing, just to be sure the cells were still there (since I've had trouble finding cells previously).
File:2014 01 27 Cot1 Positive Control Nuc1.jpg|File:2014 01 27 Cot1 Positive Control Nuc2.jpg| |
Two nucleii stained with Draq5 (red) and Alexa 488 (Cot1 DNA, green) |
File:2014 01 27 V6S3 488.jpg| |
Nucleus stained with Draq5 (red) and Alexa 488 (V6S3, green)
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