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Protein-DNA Telomere FISH

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1020

 

Protein-DNA Telomere FISH

 

FIX AND PROTEIN STRAIN:

  1. Fix cells in 4% paraformaldhyde (diluted in PBS) for 10’ at room temp.
  2. Wash cells with PBS +0.2% Tween20
  3. Permeabilize cells with 0.5% Triton X-100 for 10’ at room temp.
  4. Wash cells with PBS +0.2% Tween20
  5. Block cells with blocking buffer for 15’ at 37°C (I use o.2% Tween20 and 10% serum)
  6. 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).
  7. Wash 2 x 5’ in PBS + 0.2% Tween20.
  8. Secondary antibody stain. Stain in blocking buffer for 45 to 60’ at 37°C.
  9. Wash 2 x 5’ in PBS + 0.2% Tween20.

     

DNA FISH:

  1. Fix cells in 4% paraformaldhyde (diluted in PBS) for 10’ at room temp.
  2. Wash cells with PBS +0.2% Tween20
  3. Wash 1 x in 2XSSC for 5’ at room temp.
  4. Treat cells with RNAaseA diluted 1:100 (stock is 10 mg/ml) in 2X SSC for 45’ at 37°C.
  5. Wash 1 x in 2XSSC for 5’ at room temp.
  6. Dehydrate the slides:
    • 2’ in 70% EtOH
    • 2’ in 80% EtOH
    • 2’ in 100% EtOH
  7. Air dry the slides
  8. 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)
  9. Incubate at room temp for 2 hours in the dark.
  10. Wash slides 2 x 15’ at room temp in 70% formamide + 10mM Tris pH 7.2
  11. Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20.
  12. 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.
  13. Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20.
  14. Dehydrate the slides:
      • 3’ in 70% EtOH
      • 3’ in 80% EtOH
      • 3’ in 100% EtOH
  15. Air dry
  16. Add mounting media and cover.

 

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