Tissue Recombinants to Study Extracellular Matrix Targeting to Basement Membranes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 298

3. Methods
3.1. Creation of Intestinal Interspecies Associations (Fig. 1)
1.

Careful planning is required to get embryos at the correct stages on the day of the
experiment. In particular, incubate fertilized eggs from white Leghorn chicken at
38 ° C in a humidified incubator 5 days before the experiment

2. Remove the 5-d-old chick embryos from the eggs (day 0 being the first day of

incubation). At the same day, 12/13 day foetal mice are removed from the
placenta after laparotomy of the anaesthetized pregnant mothers (day 0 being
the day when a vaginal plug is observed). Care must be taken while embryos are
removed out of the eggs or placenta because the intestine is still externalized at
these stages.

3. Fasten the embryos with pins on the paraffin support and add sterile 9% NaCl

solution. Dissect out the intestinal chick or mouse rudiments with forceps and
Pascheff-Wolff iris scissors (Moria Dugast SA, Paris, France) under the dissect-
ing microscope. Place the intestinal anlagen onto gelified medium with the help
of a curet (3-mm diameter) and a needle-mounted probe. Remove vessels and
adherent tissues with the iris scissor.

4. Incubate the embryonic intestines in collagenase solution (about 6-10 rudiments/

per 2 mL dish) at 37 C for 1 h in a humidified incubator (5% CO 2 , 95% air) to disrupt

the basement membrane that separates the endoderm from the mesenchyme (12) .

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5. Then transfer the intestines into a dish containing the blocking solution for at

least 30 min at room temperature to stop the action of collagenase.

6. Place the intestines on gelified medium and open the mesenchymal tubes length-

wise with a microscalpel under a dissecting microscope; the endoderm can then
be pushed out of the mesenchymal gutter with forceps.

7. Cut the mesenchymes into small fragments ( 1-2 mm length) and transfer them

with the help of the curet and of the needle-mounted probe onto a dish containing
the enriched gelified medium.

8. Place in each individual mouse mesenchymal segment an equally long endoderm

derived from the chick intestine. Cover with a second mouse mesenchymal
segment. In parallel, the inverse associations are performed in between chick
mesenchymes and mouse endoderms. Control chick or mouse intestinal segments
are prepared by reassociating endoderm of each species with its own mesenchyme
( see Note 2 ).

9. Label the interspecies mesenchymal/endodermal recombinants and control

segments with a few carbon particles to identify grafted tissue; incubate over-
night in a humidified incubator (5% CO 2 , 95% air) at 37 ° C.

3.2. Grafting Procedure: Intracoelomic Grafting (Fig. 2)
1.

3-d-old chick embryos are used for grafting experiments. Put a mark on the upper
part of the shell to check that the egg is maintained in the correct position through-
out the experiment. Incubate the fertilized eggs at 38 C in the humidified incuba-

tor in an horizontal position. The day before grafting, make a hole at the sharp

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