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Stewart Laboratory Standard Operating Procedure

Stewart Laboratory Standard Operating Procedure               


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 37C (I use o.2% Tween20 and 10% serum)
  6. Stain cells with antibody of interest diluted in the blocking buffer for 60 at 37C. (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 37C.
  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 37C.
  5. Wash 1 x in 2XSSC for 5 at room temp.
  6. Dehydrate the slides:
  7. Air dry the slides
  8. Set up hybridization mix:
  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:
  15. Air dry
  16. Add mounting media and cover.