16 Whole Mount in Situ Hybridization for mRNA Detection in Chick Embryos
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- Solution A: 0.2M NaH2P04 (Store at room temperature
(RT)
- Solution B: 0.2M Na2HP04 (Store at RT)
- PBT (store at RT)
- 0.1% Tween-20 in PBS
Note: Do not autoclave after adding Tween-20.
4% (w/v) Paraformaldehyde in PBS (PFA; freshly prepared)
- 20% PFA stock in PBS (store at 4°C).
Note: Use an electron microscopy grade PFA. Heat at 65°C and
add a few drops of 5N NaOH solution with occasional agitation
until dissolved, and coolon ice. It should be stored at 4°C in
tightly closed bottles and discarded after a few weeks.
- Ethanol series (store at RT)
- 25% ethanol in PBT
- 50% ethanol in PBT
- 75% ethanol in PBT
Note: Do not use when precipitates appear.
- Ethanol (190-proof)
- 2-20~g/ml Proteinase K (freshly prepared),
- 20mg/ml stock in sterile dH20
Note: Proteinase K powder is available from Boehringer. After
dissolving in dH20, store in aliquots at -20°e.
- 2mg/ml Glycine/PBT (freshly prepared; optional)
- 0.2% Glutaraldehyde (GTA), 4% PFA/PBT (freshly prepared)
- 20% GT A stock solution (electron microscopy fixative
grade; store at 4°C)
- 20% PFA stock solution (store at 4°C)
- 6% H20 2/PBT (freshly prepared; optional)
- 30% H20 2 (store at 4°C)
- Prehybridization solution (store at -20°C)
- 50% formamide
- 5x SSC (pH4.5)
- 1% SDS
- 50~g/ml yeast tRNA
- 50llg/ml heparin
- 20x SSC (pH 4.5)