Daniel:Notebook/HiResChrPaint/2013-10-1

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FISH[edit]

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Slide Preparation[edit]

Prepping slides from yesterday. Since I'm not ready to do FISH on them quite yet, I'll just fix them for now.

Protocol[edit]

1. Rinse slides briefly in 1X PBS
2. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS
3. Rinse briefly with 1 mL 1X PBS
4. Incubate for 5 minutes in 1 mL 2X SSCT
5. Incubate for 5 minutes in 1 mL 2X SSCT + 50% (v/v) formamide
6. Aspirate, and add 1mL of 2X SSCT + 50% (v/v) formamide for storage at 4C
7. Slides are typically best used within 1-2 weeks of creation

Hybridization[edit]

Doing this on the slides from 9/24/2013

Samples[edit]

Protocol[edit]

Day 1

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; prewarm formamide
3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes
4. Remove media and allow slides to cool to room temperature
5. Add 25 uL hybridization cocktail to center of dish
6. Apply a 22x22 #1.5 cover slip to center of dish; seal with rubber cement
7. Allow the rubber cement to air-dry for 5 minutes at room temperature
8. Denature for 2.5 minutes at 92C; keep humid
9. Transfer slides to a humidified chamber and hybridize overnight at 42C