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










