16 Whole Mount in Situ Hybridization for mRNA Detection in Chick Embryos
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- 100mM Tris HCI (pH 9.5)
- 100mM NaCl
- 50mM MgCh
- 1% Tween-20
- 5% polyvinyl alcohol (PV A; 70-100 kDa; Sigma)
Note: Dissolve PV A in a microwave oven. After cooling down at
RT, add MgCh and Tween-20, and mix up to the final volume
with dH20.
- Nitroblue tetrazolium chloride (NBT) stock solution (store in
dark at -20°e)
- 75mg/ml NBT (Boehringer) in 70% dimethylformamide
(DMF)
- X-phosphate (BCIP) stock solution (store in dark at -20°e)
- 50mg/ml BCIP (Boehringer) in DMF
- Color reaction solution (freshly prepared)
- 4.5f.l1 75mg/ml NBT
- 3.5f.l1 50mg/ml BCIP
- 1ml AP buffer
- Filter
- 25%, 50% Glycerol/PBT
Procedure
1. Mix these reagents in the following order at RT:
dHzO (RNase-free)
5 x transcription buffer
O.IM dithiothreitol (DTT)l
DIG RNA Labeling Mix, 10 x cone.
Linearized template DNA z (lgll)
RNase inhibitor
RNA polymerase
Total volume is
8~1
4~1
2~1
2~1
1~1 (l~g)
l~l (20-40U)
2~1 (20-40U)
20~1
Preparation of
labeled RNA
probe