Daniel:Notebook/HiResChrPaint/2013-9-27: Difference between revisions

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Using the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|Tuesday]], I'm going to do another FISH experiment.
Using the slides from [[Daniel:Notebook/HiResChrPaint/2013-9-24#Fibroblast Adherence|Tuesday]], I'm going to do another FISH experiment.
==FISH Protocol==
*Following [[Editing Daniel:Protocols/FISH#Hybridization|standard FISH procedure]]


''Day 1''
''Day 1''
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  3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes
  3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes
  4. Remove slides and allow to cool to room temperature
  4. Remove slides and allow to cool to room temperature
  5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip
  5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip (¿add to center?)
  6. Invert slides on to cocktail-covered coverslips; seal with rubber cement
  6. Invert slides on to cocktail-covered coverslips; seal with rubber cement (¿Seal dish with parafilm?
  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 37C or 42C
  9. Transfer slides to a humidified chamber and hybridize overnight at 37C or 42C

Revision as of 18:36, 27 September 2013

FISH

Back to Calendar

Using the slides from Tuesday, I'm going to do another FISH experiment.

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 in a pre-warmed coplin jar
3. Incubate in coplin jar containing 2X SSCT + 50% formamide at 60C for 20 minutes
4. Remove slides and allow to cool to room temperature
5. Add 25 uL hybridization cocktail to a 22x22 #1.5 coverslip (¿add to center?)
6. Invert slides on to cocktail-covered coverslips; seal with rubber cement (¿Seal dish with parafilm?
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 37C or 42C