Daniel:Notebook/HiResChrPaint/2013-10-1: Difference between revisions
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>Djacobse (→FISH) |
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==Hybridization== | ==Hybridization== | ||
Doing this on the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|9/24/2013]] | |||
*Modified from [[Daniel:Protocols/FISH#Hybridization|standard FISH protocol]] | *Modified from [[Daniel:Protocols/FISH#Hybridization|standard FISH protocol]] |
Revision as of 20:40, 1 October 2013
FISH
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
- Modified from standard FISH protocol, starting at step 5
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
- Modified from standard FISH protocol
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