434
HElKE STEILEN-GIMBEL and STEFFI URBSCHAT
Outline
Immunostaining
Antigen retrieval with microwaves
Incubation with first antibody e.g. MIB-1, D07 or p16
Detection with goat-anti-mouse fluorochrome1
Two Colour FISH
Fixation
Digestion
Denaturation
Hybridization with two probes e.g. one single copy
and one a-satellite probe
Detection with streptavidin-fluorochrome2 and antiDIG-fluorochrome3
Materials
- Citrate buffer (O.OlM citric acid-monohydrate, pH 6.0)
- 4% paraformaldehyde solution (diluted in PBS), fresWy prepared
- Deionized formamide
- Hybridization mixture:
- 50% deionized formamide
- 2xSSC
- 50 mM sodium phosphate buffer pH 7.0
- 10% dextran sulfate
- Store at -20°C