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HARALD RIEDER et al.
Outline
Essential steps working with FISH on formalin-fIxed paraffIn-embedded
tissue sections are shown in the following scheme:
First Day:
Second Day:
Third Day:
Preparing specimen
Hybridization
Post-hybridization
slides
---+ procedure
f---+ procedure
Cutting sections
Deparaffinizing
Post-hybridization
Mounting sections
sections
washing procedure
Pretreating sections
Counterstaining
Fixing sections
Analysis
Probe preparation
Hybridization
Materials
First day: preparing specimen slides
Equipment - Microtome (e.g. Leitz Type 530-492)
- Water bath (e.g. GFL Type 1052)
Supplies - Positively charged slides (e.g. Super Frost/Plus Menzel) or coated slides
(poly-I-lysine, silane)
Specimens - ParaffIn blocks of conventionally embedded tissue
Second day: hybridization procedure
Equipment - Two water bath circulators (for simultaneous use), one heating up to
100
D C (e.g. RM 6 Lauda GFL 20 l)
- Heating plate, heating up to 50
D C
- Shaker (e.g. Vibrax, IKA Labortechnik)
- Microwave with temperature sensor (can be used facultatively for alternative wash procedure)
Supplies - Coverslips, 22 x 22 mm and 22 x 60 mm
- Rubber Cement (e.g. Fixogum, Marabu)
- Heat resistant Coplin jars