232
HIDEYO OHUCHI AND SUMIHARE NOJI
Note: The embryos can be stored at 4 °C at this point. The overnight wash may give lower backgrounds.
3. Wash twice with NTMT for 5 mins.
4. Wash twice with 1:1 NTMT: AP buffer for 10 mins.
5. Wash six times with AP buffer for 20 mins.
6. Incubate in the dark with color reaction solution (NBT/BCIP/
AP buffer) with gentle rocking for the first 5 mins. Periodically
monitor the reaction after the first 30 mins.
Note: It is not advisable to examine too often under the microscope because the lights will oxidize the substrate and may give
backgrounds.
7. When a strong signal is produced and/or excessive background is observed, stop the color reaction by washing several
times with NTMT.
8. Wash 5 times in PBT for 5 mins. Stained embryos can be
stored in PBT or in 50% glycerol/PBT at 4°C.
Note: The color reaction product is converted to a blue color by
washing in PBT. The embryos can be cleared in 50% glycerol/PBT.
The simultaneous detection of different target mRNAs can be
done by using probes labeled with different haptens (e.g., antifluorescein) and different substrates for color reaction (e.g., Fast
Red; Dietrich et al. 1997).
Destaining
If the embryos are overstained, it is possible to partially destain
them in ethanol, but care has to be taken in case ethanol removes
too much signal or by brief incubation in ethanol.
1. Incubate in PBT for 1hr at RT.
2. Wash with 50% ethanol in PBT for 5 mins.
3. Wash with 100% ethanol (190-proof) for up to 2hrs.
4. Wash with 50% ethanol in PBT for 5 mins.
5. Wash with PBT for 5 mins.
HIDEYO OHUCHI AND SUMIHARE NOJI
Note: The embryos can be stored at 4 °C at this point. The overnight wash may give lower backgrounds.
3. Wash twice with NTMT for 5 mins.
4. Wash twice with 1:1 NTMT: AP buffer for 10 mins.
5. Wash six times with AP buffer for 20 mins.
6. Incubate in the dark with color reaction solution (NBT/BCIP/
AP buffer) with gentle rocking for the first 5 mins. Periodically
monitor the reaction after the first 30 mins.
Note: It is not advisable to examine too often under the microscope because the lights will oxidize the substrate and may give
backgrounds.
7. When a strong signal is produced and/or excessive background is observed, stop the color reaction by washing several
times with NTMT.
8. Wash 5 times in PBT for 5 mins. Stained embryos can be
stored in PBT or in 50% glycerol/PBT at 4°C.
Note: The color reaction product is converted to a blue color by
washing in PBT. The embryos can be cleared in 50% glycerol/PBT.
The simultaneous detection of different target mRNAs can be
done by using probes labeled with different haptens (e.g., antifluorescein) and different substrates for color reaction (e.g., Fast
Red; Dietrich et al. 1997).
Destaining
If the embryos are overstained, it is possible to partially destain
them in ethanol, but care has to be taken in case ethanol removes
too much signal or by brief incubation in ethanol.
1. Incubate in PBT for 1hr at RT.
2. Wash with 50% ethanol in PBT for 5 mins.
3. Wash with 100% ethanol (190-proof) for up to 2hrs.
4. Wash with 50% ethanol in PBT for 5 mins.
5. Wash with PBT for 5 mins.