• Useful for samples that have been fixed for extended periods of time, or those that are difficult to use as FISH targets, such as brain samples.
• Deparaffinize a 40 µm tissue section with xylene (or Histoclear) 3x 10 minutes each.
• Dehydrate in 100% ETOH 2 x 5 minutes each.
• Incubate in 4 mg/ml pepsin (Sigma P-7012, in 0.9% NaCl pH, 1.5), 2 hrs. at 37 C. Vortex every 30 min.
• Filter through 40 µm nylon mesh.
• Spin at 800 rpm (standard tabletop) 5-10 minutes.
• Wash pellet in PBS, spin 2 x 800 rpm 5-10 minutes each.
• Resuspend pellet in a minimal volume of PBS.
• Vortex nuclei, spread on FISHer Superfrost Plus (cat.# 12-550-15) slides, dry slides at 65 C for 10 minutes.
• Rinse slide in PBS.
• Incubate in 0.01% Triton 100 in PBS for 1.5 minutes.
• Rinse 3 x in PBS, 3 minutes each.
• Incubate 5 minutes in 0.3 mg/ml pronase in 50 mM Tris/Cl pH 7.6, 5mM EDTA.
• Incubate in 2 mg/ml glycine in PBS 3 x 3 minutes each.
• Incubate 5 minutes in 4% paraformaldehyde in PBS.
• Incubate 3 minutes in 2 mg/ml glycine in PBS.
• Wash in 30%, 60%, 80%, 95% and 100% ETOH for 3 minutes each.
• Air dry slides.
• Denature probe and target DNA simultaneously for 1-3 minutes in a 90 C oven.
• Incubate at 37 C in a humidified chamber at least 1 hour to overnight.
• Wash for 1 minute each in the following wash solutions at 45 C: 3 x 50% formamide/2XSSC, one wash each in 2X SSC, 2X SSC/0.1% NP-40, 2X SSC/0.1% NP-40 (RT).
• This protocol calls for formamide and xylene, which are teratogens. Refer to the respective MSDS for more information.

Protocol submitted by:

Dr. Rizwan Naaem - Director - Cytogenetics Lab

Baystate Medical Center, Springfield, MA 1994