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STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
• Pepsin ( e.g. from porcine stomach mucosa, 500 - 3000 units/mg).
• Digestion solution: 1-2 mg pepsin/100 ml in 0.01 M HCL
• 0.5% Paraformaldehyde-PBS-solution (freshly prepared).
Procedure
ln advance 0.5% Paraformaldehyde-PBS-solution: In a fume hood (!) dissolve paraformaldehydein PBS/5mM MgCh, heat at 80°C, adjust to pH 7 using 1M NaOH.
Under these conditions, most of the paraformaldehyde solution is dissolved; the remainder is removed by paper f:tltration.
Pepsin digestion 1. Wash slides containing f:txed chromosomes in PBS for 5 min at 37°C.
2. Incubate in digestion solution for 10 min at 37°C.
3. Wash slides in PBS for 5 min at room temperature.
4. Immerse slides in 0.5% paraformaldehyde-PBS-solution for 5 min at
room temperature.
5. W ash slides 3 tim es in PBS for 5 min each.
6. Dehydrate slides in a series of 70%, 90%, 100% ethanol 5 min each.
7. Proceed with fluorescence in situ hybridization protocol (see Chap. 12).
Subprotocol 4
Preparation of Fibroblasts for FISH (Adherently Growing Cells)
Subprotocol2 can also be applied for the pretreatment of adherently growing cells. There are some minor modif:tcations, however, that are described
in the following for the preparation of human f:tbroblasts.
Materials
• Colcemid solution: prepare stock solution of 10 f.!g/ml in water and store
at -20°C.
• Trypsin-EDTA solution: 2.5 g trypsin and 0.38 g EDTA dissolved in 1000
ml PBS (-Mg2+, -Ca 2 +).
• Fetal calf serum (FCS).
STEFAN JOOS, MARTIN BENTZ, ANTON H. N. HOPMAN AND PETER LICHTER
• Pepsin ( e.g. from porcine stomach mucosa, 500 - 3000 units/mg).
• Digestion solution: 1-2 mg pepsin/100 ml in 0.01 M HCL
• 0.5% Paraformaldehyde-PBS-solution (freshly prepared).
Procedure
ln advance 0.5% Paraformaldehyde-PBS-solution: In a fume hood (!) dissolve paraformaldehydein PBS/5mM MgCh, heat at 80°C, adjust to pH 7 using 1M NaOH.
Under these conditions, most of the paraformaldehyde solution is dissolved; the remainder is removed by paper f:tltration.
Pepsin digestion 1. Wash slides containing f:txed chromosomes in PBS for 5 min at 37°C.
2. Incubate in digestion solution for 10 min at 37°C.
3. Wash slides in PBS for 5 min at room temperature.
4. Immerse slides in 0.5% paraformaldehyde-PBS-solution for 5 min at
room temperature.
5. W ash slides 3 tim es in PBS for 5 min each.
6. Dehydrate slides in a series of 70%, 90%, 100% ethanol 5 min each.
7. Proceed with fluorescence in situ hybridization protocol (see Chap. 12).
Subprotocol 4
Preparation of Fibroblasts for FISH (Adherently Growing Cells)
Subprotocol2 can also be applied for the pretreatment of adherently growing cells. There are some minor modif:tcations, however, that are described
in the following for the preparation of human f:tbroblasts.
Materials
• Colcemid solution: prepare stock solution of 10 f.!g/ml in water and store
at -20°C.
• Trypsin-EDTA solution: 2.5 g trypsin and 0.38 g EDTA dissolved in 1000
ml PBS (-Mg2+, -Ca 2 +).
• Fetal calf serum (FCS).