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 |
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