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