Daniel:Notebook/HiResChrPaint/2013-10-1: Difference between revisions
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Doing this on the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|9/24/2013]] | Doing this on the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|9/24/2013]] | ||
===Samples=== | |||
*'''Experiment''': 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-8-5#Labeling Results|V6S1]], 10 pmol [[Daniel:Notebook/HiResChrPaint/2013-7-17#Labeling Results|V6S3]] | |||
*'''Control''': 20 pmol [[Daniel:Notebook/HiResChrPaint/2013-9-5#Labeling Results|BSPP-2]] | |||
===Protocol=== | |||
*Modified from [[Daniel:Protocols/FISH#Hybridization|standard FISH protocol]] | *Modified from [[Daniel:Protocols/FISH#Hybridization|standard FISH protocol]] | ||
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3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes | 3. Incubate in 2X SSCT + 50% formamide at 60C for 20 minutes | ||
4. Remove media and allow slides to cool to room temperature | 4. Remove media and allow slides to cool to room temperature | ||
5. Add 25 uL hybridization cocktail to a 22x22 #1.5 | 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 | 7. Allow the rubber cement to air-dry for 5 minutes at room temperature | ||
8. Denature for 2.5 minutes at 92C; keep humid | 8. Denature for 2.5 minutes at 92C; keep humid | ||
9. Transfer slides to a humidified chamber and hybridize overnight at 42C | 9. Transfer slides to a humidified chamber and hybridize overnight at 42C |
Latest revision as of 22:05, 1 October 2013
FISH[edit]
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]
- 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[edit]
Doing this on the slides from 9/24/2013
Samples[edit]
Protocol[edit]
- 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 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