11 Specimen Preparation Techniques for Cytogenetic Analysis by Fluorescence in situ Hybridization
161
5. Briefly wash slides 5 times in water then 5 times in PBS.
6. Fix the cells in 1 o/o paraformaldehyde (dissolved in 0.1 M phosphate buffer) for 10 min at room temperature.
7. Briefly wash slides 5 times in water then 5 times in PBS.
8. Dehydrate slides in 70%, 90% and 100% ethanol for 5 min each, then air
dry.
9. Proceed as described in Subprotocol 8 (step 10-12).
Subprotocol 10
Paraffin-Embedded Tissue Sections
Materials
• 0.2 M HCl
• 1 o/o H20 2 dissolved in methanol
• Xylene
• Methanol
• Phosphate-buffered saline (PBS)
• 1 M sodium thiocyanate (N aSCN)
• Digestion solution: Pepsin from porcine stomach mucosa (2500 - 3000
units/mg). Dissolve about 4 mg pepsin/ml in 0.2 M HCl
• Denaturation solution: 70% deionized formamide/2x SSC/SOmM sodium phosphate, pH 7
• 70%, 90% and 100% ethanol
• Hybridization solution: 50% deionized formamide/2x SSC/10% dextran
sulfate
Procedure
Glutaraldehyde-activated, poly-L-lysine coated slides: Coat the slides as de- ln advance
scribed in Subprotocol 7 and activate the poly-L-lysine coated slides with