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

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

Cross-references are to Schwarzacher & Heslop-Harrison 2000. Practical in situ Hybridization. Bios, Oxford. 216+xii pp . Available from Sigma Chemical

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
 
See Castilho et al. J. Cell Sci. 2000 (on Publication page)

Pat Heslop-Harrison