160
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Preparation of
frozen sections
Note: Small tumor samples ( cytological specimens) can be placed directly in
a tube containing digestion solution including pepsin before the cells are
put on slides.
Note: Denaturation of the probe as well as the target DNA can be performed
in a single step: After adding the probe to the specimen, a cover slip is applied and sealed with ruhher cement. The slide is heated for about 3 min at
70°C and then transferred to 37°C.
Subprotocol 9
Preparation of Frazen Sections
Materials
• Poly-L-lysine coated slides
• Digestion solution: Pepsin from porcine stomach mucosa (2500 - 3000
units/mg). Dissolve 50-400 ~g pepsin/ml in 0.01 M HCl
• 1% paraformaldehyde, dissolved in 0.1 M phosphate buffer (pH 7.2-7.4)
containing 5 mM MgC12•
• Methanol-acetone (1:1)
• 70%, 90% and 100% ethanol series.
• 70% ethanol ( -20°C)
• Phosphate-buffered saline (PBS)
• Tween-20
Procedure
1. Mount frozen tissue section on poly-L-lysine coated slides and airdry at
room temperature.
2. Fix sections in methanol-acetone (1:1) for 20 min at -20°C.
3. Wash slides twice in PBS containing 0.5% Tween-20 for 5 min each.
4. Incubate slides for 10 min in 200 ~1 of digestion solution at 37°C. It may
be necessary to modify the incubation time empirically.
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
Preparation of
frozen sections
Note: Small tumor samples ( cytological specimens) can be placed directly in
a tube containing digestion solution including pepsin before the cells are
put on slides.
Note: Denaturation of the probe as well as the target DNA can be performed
in a single step: After adding the probe to the specimen, a cover slip is applied and sealed with ruhher cement. The slide is heated for about 3 min at
70°C and then transferred to 37°C.
Subprotocol 9
Preparation of Frazen Sections
Materials
• Poly-L-lysine coated slides
• Digestion solution: Pepsin from porcine stomach mucosa (2500 - 3000
units/mg). Dissolve 50-400 ~g pepsin/ml in 0.01 M HCl
• 1% paraformaldehyde, dissolved in 0.1 M phosphate buffer (pH 7.2-7.4)
containing 5 mM MgC12•
• Methanol-acetone (1:1)
• 70%, 90% and 100% ethanol series.
• 70% ethanol ( -20°C)
• Phosphate-buffered saline (PBS)
• Tween-20
Procedure
1. Mount frozen tissue section on poly-L-lysine coated slides and airdry at
room temperature.
2. Fix sections in methanol-acetone (1:1) for 20 min at -20°C.
3. Wash slides twice in PBS containing 0.5% Tween-20 for 5 min each.
4. Incubate slides for 10 min in 200 ~1 of digestion solution at 37°C. It may
be necessary to modify the incubation time empirically.