Protein-DNA Telomere FISH
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Protein-DNA Telomere FISH
FIX AND PROTEIN STRAIN:
- Fix cells in 4% paraformaldhyde (diluted in PBS) for 10’ at room temp.
- Wash cells with PBS +0.2% Tween20
- Permeabilize cells with 0.5% Triton X-100 for 10’ at room temp.
- Wash cells with PBS +0.2% Tween20
- Block cells with blocking buffer for 15’ at 37°C (I use o.2% Tween20 and 10% serum)
- Stain cells with antibody of interest diluted in the blocking buffer for 60’ at 37°C. (I usually use dilution ranging from 1:100 to 1:500).
- Wash 2 x 5’ in PBS + 0.2% Tween20.
- Secondary antibody stain. Stain in blocking buffer for 45 to 60’ at 37°C.
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Wash 2 x 5’ in PBS + 0.2% Tween20.
DNA FISH:
- Fix cells in 4% paraformaldhyde (diluted in PBS) for 10’ at room temp.
- Wash cells with PBS +0.2% Tween20
- Wash 1 x in 2XSSC for 5’ at room temp.
- Treat cells with RNAaseA diluted 1:100 (stock is 10 mg/ml) in 2X SSC for 45’ at 37°C.
- Wash 1 x in 2XSSC for 5’ at room temp.
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Dehydrate the slides:
- 2’ in 70% EtOH
- 2’ in 80% EtOH
- 2’ in 100% EtOH
- Air dry the slides
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Set up hybridization mix:
- Make blocking buffer (Roche 1096176, must be made up in 0.1M maleic acid and 150 mM NaCl, raise temp to 50-60°C and stir to get into solution).
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Set up hyb: 70% Formamide
- 0.3 μg/ml probe (if you use my aliquots you should add 5 mls to the tubes to get this concentration)
- 1% blocking buffer
- 10 mM Tris pH 7.2 (I often use 7.0)
- Incubate at room temp for 2 hours in the dark.
- Wash slides 2 x 15’ at room temp in 70% formamide + 10mM Tris pH 7.2
- Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20.
- Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20 + 0.1 ug.ml DAPI.
- Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20.
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Dehydrate the slides:
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- 3’ in 70% EtOH
- 3’ in 80% EtOH
- 3’ in 100% EtOH
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- Air dry
- Add mounting media and cover.