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In situ immunodetection of 5-methylcytosine (5-mC) on squashed cells using anti-methylcytosine

 

  1. Prepare chromosomes as for in situ hybridization (Chapter 5 in situ book)
  2. Slides are pre-treated with RNase and pepsin (Protocol 8.1 in situ book without paraformaldehyde treatment)
  3. Ethanol dehydrate the slides and air dry
  4. Block slides in PBST + 1% BSA for 20 min at RT
  5. Incubate slides in monoclonal anti-5-mC (1:1000 dilution in PBS) for 1h at 37oC in a humid chamber
  6. Wash in PBST 2x5min
  7. Incubate slides in Biotin anti-mouse IgG (1:500 dilution in PBST) for 1h at 37oC in a humid chamber
  8. Wash in PBST 2x5min
  9. Incubate slides in streptavidin Cy3 (1:300 dilution in PBST) for 1h at 37oC in a humid chamber
  10. Wash in PBST 2x5min
  11. DAPI stain and mount (Protocol 9.3 in situ book)

Solutions:
PBS (see Appendix 1 in situ book)
PBST: add 0.03% (v/v) Tween 20 to the 1xPBS