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Home  |  Protocols  |  In situ hybridization and Immunochemistry  |  Whole mount in situ hybridizatyon on mouse embryos with DIG-and FLUO-labeled riboprobes
Home  |  Protocols  |  In situ hybridization and Immunochemistry  |  Whole mount in situ hybridizatyon on mouse embryos with DIG-and FLUO-labeled riboprobes

Protocol - Whole mount in situ hybridizatyon on mouse embryos with DIG-and FLUO-labeled riboprobes

It is used to detect expression of specific markers in mouse embryos

Whole mount in situ hybridizatyon on mouse embryos with DIG-and FLUO-labeled riboprobes
Category:  In situ hybridization and Immunochemistry
Last revision: Nov 26, 2014
Contact
Name: Giovanna L. Liguori
Address: Institute of Genetics and Biophysics A. Buzzati-Traverso - CNR, Via P. Castellino 111, 80131 Naples, Italy
Email: giovanna.liguori@igb.cnr.it

 

Figure legend:

On the left expression of  Lefty2 (blue) and Otx2 (red) genes in 7.5 dpc mouse mbryo; on the right  expression of Wnt1 (blue) and Shh (red) genes in 8.5 dpc mouse embryo

Steps

Description Temperature Time Note
Rehydrate embryos: 75, 50, 25% methanol:PBT
RT
5min
Wash 2x in PBT
RT
5min
Bleach 6% hydrogen peroxide (H2O2)in PBT
RT
1h
Wash 3x in PBT
RT
5min
Incubate in 10 μg/ml proteinase K in PBT
RT
6 min for 5.5-6.5 dpc; 8 min for 7.5-8.5 dpc; 10 min for explants 9.5 dpc 12 min for 9.5 dpc and older
proteinase K 10mg/ml (1000X) in water, stored at -20°C
Wash 1x in PBT
RT
few seconds
Wash 2x in PBT
RT
5min
Refix 0.2% gluteraldehyde/4% PFA in PBT
RT
20 min
Wash 2x PBT
RT
5min
Prehybridize in the hybridization mix
68°C
2-3 h
The embryos can be saved in hybridizatyon solution at -20°C
Hybridize
68°C
O.N.
probe concentration 100-300 ng/ml
Wash 3x Sol I
68°C
45min
Wash 3x Sol II
68°C
45min
Wash 3x MABT1X
RT
5 min
Preblock in 10% sheep serum in MABT 1X
RT
about 3h
rocking
Incubate with Ab anti-DIG
4°C
O.N.
anti-DIG (1:3000) in 1% sheep serum /MABT 1X. Rocking
Wash 6x MABT1X
RT
1h
rocking
Wash with MABT1X
4°C
O.N.
rocking (facoltative)
Wash 3x NTMT1X
RT
10min
Incubate in dark
0.5 μl NBT/ml e 3.5 μl BCIP/ml NTMT 1X
Stop the reaction with several washes in PBT 1X
Dehydrate embryos: 25, 50, 75, 100% methanol:PBT
RT
10min
Rehydrate embryos: 100, 75, 50, 25% methanol:PBT
RT
10min
Wash 2x in PBT1X
RT
5min
or save in PBT1X
Wash 3x MABT1X
RT
5min
Preblock in 10% sheep serum in MABT 1X
about 2h
Incubate with Ab anti-FLUO
4°C
O.N
anti-FLUO (1:4000) in 1% sheep serum/ MABT 1X. Rocking
Wash 6x MABT1X
RT
60min
Wash with MABT1X
4°C
O.N.
rocking (facultative)
Wash 3x NTMT1X
RT
10min
Incubate in dark
7μl INT-BCIP/ml NTMT 1X
Stop the reaction in PBT 1X.
Save
4°C

Other informations

The protocol was provided in 2001 by Dr. Juan Pedro Martinez Barbera, used in Dr. Rosa Beddington laboratory (National Institute for Medical Research, London, UK).

For the washes use:
- 12-multiwell plates
- Millipore baskets

Consider for each sample a V of 2ml/wash

 

Quality validation: Yes

Validation info

The protocol has been used  to produce most of the data of several articles published  in peer-reviewed journals.

 

Citations
Liguori G.L., Echevarria D., Bonilla S., D’Andrea D., Liguoro A., Persico M.G., Martinez S. Characterization of the functional Properties of the neuroectoderm in mouse Cripto-/- embryos showing severe gastrulation defects. Int J Dev Biol. 2009;53:549-557.
Di Palma T., D’Andrea B., Liguori G.L., Liguoro A., de Cristofaro T., Del Prete D. Pappalardo A., Zannini M. TAZ is a critical coactivator for Pax8 and TTF-1, two transcription factors necessary for thyroid differentiation. Exp Cell Res.2009;315:162-175.
D’Andrea D., Liguori G.L., Le Good A.J., Lonardo E., Andersson O., Constam D.B., Persico M.G., Minchiotti G. Cripto promotes A-P axis specification independently of its stimulatory effect on Nodal auto-induction. J Cell Biol 2008;180:597-605.
Liguori G.L., Borges, A.C., D’Andrea D., Liguoro A., Gonçalves L., Salgueiro A.M., Persico M.G and Belo J.A. A Cripto-independent nodal signalling promotes positioning of the A-P axis in the early mouse embryo. Dev Biol 2008;315;280-289.
Liguori G.L., Echevarria, D., Improta, R., Signore, M., Adamson, E., Martinez, S., Persico, M.G. Anterior neural plate regionalization in cripto null mutant mouse embryos in the absence of node and primitive streak. Dev Biol 2003;264:537-549.

 

Related Protocols:
Related ModelSystems:

Solutions

  • PBT (500ml)

    Reagents F-concentration Quantity I-concentration
    PBS
    1X
    50ml
    10X
    Tween-20
    0.1%
    5ml
    10%

  • 6% H2O2 in PBT (10ml)

    Reagents F-concentration Quantity I-concentration
    H2O2
    6%
    2ml
    30% (4°C)
    PBT
    1X
    8ml
    1X

  • 0.2% glut / 4% PFA in PBT (10ml)

    Reagents F-concentration Quantity I-concentration
    PFA
    4%
    2ml
    20% (-20°C)
    glut
    0.2%
    80μl
    25% (-20°C)
    PBT
    1X
    8ml
    1X

  • Hybridization Mix (100ml)

    Reagents F-concentration Quantity I-concentration
    Formamide
    50%
    50ml
    100%
    SSC pH 4.5-5
    5X
    25ml
    20X
    Tween 20
    0.1%
    1ml
    10%
    Heparin
    50 μg/ml
    0.1 ml
    50 mg/ml
    Add at the moment only in the solution you have to use
    tRNA
    50 μg/ml
    10 mg/ml
    DNA salmon sperm
    50 μg/ml
    10 mg/ml

    Note

    Store hybridization mix (w/o DNAss and tRNA) at -20°C.

    Add DNAss and tRNA just before using the hybridization solution.

  • Sol I (10ml)

    Reagents F-concentration Quantity I-concentration
    Formamide
    50%
    5ml
    100%
    SSC pH 4.5-5
    4X
    2 ml
    20X
    SDS
    1%
    0.5 ml
    20%

  • Sol II (10ml)

    Reagents F-concentration Quantity I-concentration
    Formamide
    50%
    5ml
    100%
    SSC pH 4.5-5
    2X
    1ml
    20X

  • MAB 5X pH7.5 (400ml)

    Reagents F-concentration Quantity I-concentration
    Maleic acid
    0.5M
    23.2g
    NaCl 0.75M
    0.75M
    17.4g

    Note

    Wait that all the maleic acid is melt and later add NaCl. Reach the adequate pH adding NaOH

  • MABT 1X (50ml)

    Reagents F-concentration Quantity I-concentration
    MAB
    1X
    10ml
    5X
    Tween 20
    0.1%
    0.5ml
    10%

  • NTMT 1X (50ml)

    Reagents F-concentration Quantity I-concentration
    NaCl
    0.1M
    1ml
    5M
    Tris-HCl pH 9.5
    0.1M
    5ml
    1M
    MgCl2
    0.05M
    2.5ml
    1M
    Tween 20
    0.1%
    0.5ml
    10%

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