Fluorescence In Situ Hybridization

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

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