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PRE-TREATMENT

Fix the cells with Carnoy’s and drop them onto a glass slide Air dry (and store in -20 oC until use) Age (if cells are fresh) the cells in 2xSSC, 30 min, 37 oC Treat with 10-50 μg/mL pepsin in 0.01 N HCl at 37oC, 1-5 min Wash PBS/0.5 M MgCl2, 5 min, twice Postfix with 1% formaldehyde/PBS-Mg l2, 5 min Wash PBS, 5 min Dehydrate slide in 70%, 80%, 100% EtOH, 2 min each Air Dry 5 min Denature slide at 76oC in 70% Formamide/2xSSC, 5 min Dehydrate slide: ice cold 70% EtOH, 80%, 100%, 2 min each Air dry

PROBE PREPARATION

Synthesize labeled-probes using Nick Translation Kit (Vysis), Random Priming Kit, etc. Mix probes with 1 μl ssDNA, 1 μl Human Cot-1 DNA, and 7 μl MM2.1

Denature probe mixture at 76oC, 5 min Incubate at 37oC for 30-60min, competition

Optional: Co-denaturation

Apply probes to slide and denature at 80oC on a heat block for 2-5 minutes

HYBRIDIZATION

Apply probes to dried slide Seal with rubber cement Incubate at 37oC in humidified chamber, up-side-down, 1-2 days

WAHSING

Remove rubber cement Wash with 50% formamide/2xSSC at 43oC, 10 min, 3 times, coverslip falls off in 1st wash Wash with 2xSSC at 43oC, 10 min Wash 2xSSC/0.1% NP-40 at 43oC, 5 min Mount in DAPI

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Master Mix 2.1 1 g dextran sulfate 5.5 mL formamide 0.5 20X SSC x mL water ------------------------------ 7 mL total

Heat to 70oC to dissolve dextran sulfate pH to 7

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