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 37℃ 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 37℃ in a humid chamber

8) Wash in PBST 2x5min

9) Incubate slides in streptavidin Cy3 (1:300 dilution in PBST) for 1h at 37℃ 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