16 Whole Mount in Situ Hybridization for mRNA Detection in Chick Embryos
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11. Redissolve in prehybridization buffer at 0.1 - 0.5 Ilgilli. Store
at -80 a C. Use appropriate volume for hybridization mix at
100-500 ngiml.
Note: We have found it better to use long probes as they are much
more sensitive than short probes.
Fixation and dehydration of embryos
1. Dissect embryos in PBS, removing as much extraneous tissue as possible. To avoid the trapping of reagents, open any
cavities, such as the brain vesicle, optic vesicles, otic vesicles,
and heart, using a needle (27 gauge or less).
2. Fix in paraformaldehyde fixative at 4°C, 2 hrs to overnight.
3. Rinse the embryos with PBT, twice for 5 mins.
4. Dehydrate the embryos by washing for 5 mins each in a
graded ethanol series diluted in PBT (25% ethanol, 50%
ethanol, 75% ethanol) and then twice with 100% ethanol
(190-proof). The embryos can now be stored at -20°C for
at least several months.
Pretreatment and hybridization of the embryos
Note: Unless otherwise stated all treatment are done at RT.
Leave a little liquid above the embryos after each wash or you
will damage the embryos.
All washes are carried out with gentle rocking.
1. Transfer the embryos into a 24-well dish. Rehydrate by taking through the ethanol! PBT in reverse and then wash twice
with PBT.
2. Treat the embryos with 2 20llgiml proteinase K in PBT for up
to 15 mins
Note: The length and time of treatment depends on the experimental purpose (e.g., the size of tissue, the stage of embryos,
epithelial or mesenchymal tissue) and should be optimized.
We usually treat chick embryos with IOllgiml for X mins (X=
229
11. Redissolve in prehybridization buffer at 0.1 - 0.5 Ilgilli. Store
at -80 a C. Use appropriate volume for hybridization mix at
100-500 ngiml.
Note: We have found it better to use long probes as they are much
more sensitive than short probes.
Fixation and dehydration of embryos
1. Dissect embryos in PBS, removing as much extraneous tissue as possible. To avoid the trapping of reagents, open any
cavities, such as the brain vesicle, optic vesicles, otic vesicles,
and heart, using a needle (27 gauge or less).
2. Fix in paraformaldehyde fixative at 4°C, 2 hrs to overnight.
3. Rinse the embryos with PBT, twice for 5 mins.
4. Dehydrate the embryos by washing for 5 mins each in a
graded ethanol series diluted in PBT (25% ethanol, 50%
ethanol, 75% ethanol) and then twice with 100% ethanol
(190-proof). The embryos can now be stored at -20°C for
at least several months.
Pretreatment and hybridization of the embryos
Note: Unless otherwise stated all treatment are done at RT.
Leave a little liquid above the embryos after each wash or you
will damage the embryos.
All washes are carried out with gentle rocking.
1. Transfer the embryos into a 24-well dish. Rehydrate by taking through the ethanol! PBT in reverse and then wash twice
with PBT.
2. Treat the embryos with 2 20llgiml proteinase K in PBT for up
to 15 mins
Note: The length and time of treatment depends on the experimental purpose (e.g., the size of tissue, the stage of embryos,
epithelial or mesenchymal tissue) and should be optimized.
We usually treat chick embryos with IOllgiml for X mins (X=