Protocol for FISH on tissue sections for Human XY FISH probes and Rat Y FISH probe

< Pretreatment >

Deparaffinize sections
Keep sections in 2xSSC for 5 min at RT

(optional)

Heat sections in a microwave oven for 10 min in 2xSSC (check buffer level)
   Cool sections in PBS at RT

Treat sections in 0.02% – 0.5% pepsin / 0.1N HCl for 1 – 30 min
Keep sections in PBS for 5 min at RT
Dehydrate using alcohol series and dry

< Hybridization >

Apply 10 ml of probe and cover by coverslips
Denature at 80 – 90ºC for 10min 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 46ºC
Keep slides in 1xSSC for 15 min at RT

（in the case of biotin-labelled probes）

Block slides using blocking reagent（5% milk or 1% BSA in 0.1% Nonidet P-40 / 4xSSC, etc）
Incubate with fluolophore-conjugated avidin or streptaviden 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