Protocol for FISH on metaphase spreads

< Pretreatment >

If needed,
Wash slides in PBS at RT
Dehydrate using alcohol series and dry

< Hybridization >

Apply 10 μl of probe and cover by coverslips
Denature at 70ºC for 5 min using a hotplate
Hybridize overnight at 37ºC in a wet chamber

< Washing >

Keep hybridized slides in 2xSSC for 5 min and remove coverslips gently
Keep slides in 50% formamide / 2xSSC for 20min at 37ºC
Keep slides in 1xSSC for 15 min at RT

(in the case of hapten-labelled probes)

Block slides using blocking reagent(5% milk or 1% BSA in 4xSSC)
Incubate slides with fluolophore-conjugated antibody or streptavidin in blocking reagent for 30 – 60 min at 37ºC
Wash slides with 0.1% Nonidet P-40 / 4xSSC for 10 min at RT for 3 times

< Counterstain >

Stain with DAPI and mount with antifade mounting medium