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Fluorescence In Situ Hybridization

DNA FISH: Single cells on glass slide
5/22/07

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

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