Daniel:Notebook/HiResChrPaint/2013-10-1

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Revision as of 20:40, 1 October 2013 by >Djacobse (→‎Hybridization)
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FISH

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Slide Preparation

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

Protocol

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

Doing this on the slides from 9/24/2013

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 a 22x22 #1.5 coverslip
6. Invert slides on to cocktail-covered coverslips; 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