200
HARUO OHTANI
Hybridization
(RNase-free
condition)
Posthybridization washing
Add the following (for 1000/11):
20mg/ml E. coli tRNA
l.OM Tris HCI buffer, pH8.0
O.5M EDTA, pH8.0
20x Denhart's solution
SM NaCI
deionized formamide
DEPC-treated water
50% dextran sulfate
digoxigenin-Iabeled cRNA
SO!!1
20!!1
S!!l
SO!!1
60!!1
SOO!!1
lIS!!1
200!!1
an appropriate volume
Note: The final concentration of the probes is set at l/1g/ml at
first. If the reaction is too strong, dilute the probe concentration.
Vortex well. Heat the solution at 95°C for 5 mins and cool it on
ice.
Apply the hybridization solution on the glass slides. The
quantity of the hybridization solutions is 20-80/11 per one section
depending on the size of the specimens. Incubate the sections at
45°C for 16hrs.
The cover glass is not required. On those occasions, the moist
chamber used for the hybridization should contain 2x SCC/SO%
formamide (FA) to avoid drying or excessive hydration ofhybridization solution applied on the specimens.
The glassware and solutions should be RNase free till this process. It may be difficult to make the moist chamber RNase-free.
In this case, the chambers should be carefully washed each time
after use.
Use conventional glassware and solutions (no need for RNasefree procedures after this).
1. Wash in 2x SSC/SO% FA for Ih at 50°C.
Note: The temperature can be set at 4S-S0°C in the washing procedures. This should be set higher when non-specific reactivity is
observed.