Protein-DNA?Telomere?FISH

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Protein-DNA Telomere FISH

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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. Wash 2 x 5’ in PBS + 0.2% Tween20.

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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. Dehydrate the slides:

• 2’ in 70% EtOH
• 2’ in 80% EtOH
• 2’ in 100% EtOH

Air dry the slides 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).
• 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. Dehydrate the slides:

• • 3’ in 70% EtOH
  • 3’ in 80% EtOH
  • 3’ in 100% EtOH

Air dry Add mounting media and cover.

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