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Protocol - Gelatin embedding

Gelatin embedding of mouse embryos for cryosections

Category: Histology

Last revision: Mar 01, 2016

Contact

Name: Giovanna L. Liguori

Address: Institute of Genetic and Biophisic "A. Buzzati-Traverso", CNR. Via Pietro Castellino 111, 80131 Napoli

Email: giovanna.liguori@igb.cnr.it

Figure legend:

Transverse cryosection of a mouse 8.5 dpc embryo after Wnt1/Shh Double WISH

Steps

Description	Temperature	Time
Wash in PBS1X	RT	2x10 min
Transfer the embryos to 15% surcrose in PBS and leave at 4°C until the embryos sink.	4°C	some hours/O.N
Prepare 7.5% gelatin in 15% surcrose-PBS and leave to melt at 37°C until it is completely melt and with no bubbles
Put each embryo (or all the ones to include in the same mould) in a tube in about 1-2 ml of gelatin solution.	37°C	30 min.
Prepare a gelatin bed in a small Petri dish (2 ml about) and let it solidify under the microscope. When it begins to be solidified put the embryo on the top and cover with other gelatin solution. Orientate using an eyelash. Let it solidify under the microscope and then transfer at 4°C
Put isopentane (2 Met-butane) in a becker in dry ice at least 1h before use it or in N liquid
Using a bisturi knife cut a cubic portion
Put Tissuetek without bubbles on hard card and later put the gelatin cube in the wright orientation. Cover with Tissuetek
With large forceps put the cube in isopentane and keep.		1 min
Let in dry ice for some minutes
Save the samples	-80°C

This protocol can be applied on both fixed embryos (4% PFA in PBS or PBT for 3h/ON) or after the WISH (Whole-mount in situ hybridization).

The protocol was provided in 2004 to Dr. Giovanna L. Liguori by the laboratory of Dr. J.A. Belo (Instituto Gulbenkian, Oeiras, PT).

Quality validation: Yes

The protocol has been extensively used to produce data published in peer-reviewed journals

Citations
Di Palma T, D'Andrea B, Liguori GL, Liguoro A, de Cristofaro T, Del Prete D, Pappalardo A, Mascia A, Zannini M. TAZ is a coactivator for Pax8 and TTF-1, two transcription factors involved in thyroid differentiation. Exp Cell Res. 2009;315:162-75.
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.

Related Protocols:

Related ModelSystems:

Reagents	F-concentration	Quantity	I-concentration
Sucrose	15%	30g
PBS 1X		200ml