Tissue Recombinants to Study Extracellular Matrix Targeting to Basement Membranes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 296

capillary ingrowth in these interspecies chimeric kidneys using species-
specific antibodies.
To distinguish the deposition of a single molecule at the subepithelial base-
ment membrane, we designed a new experimental model using recombinants
between chick and mouse embryonic intestines. Isolation of pure intestinal
endodermal and mesenchymal compartments was performed by enzymatic and
mechanical treatments. Following constructions of interspecies endodermal/
mesenchymal associations, developmental growth was achieved by in vivo
transplantation. Immunocytochemistry using species-specific antibodies
recognizing either chick or mouse basement membrane molecules was then
performed on cryosections made through the developed hybrid intestines.
The use of this experimental design permits determination of the precise
chronological expression/deposition at the intestinal basement membrane
region of the individual constituents: some of them are strictly of epithelial or
mesenchymal origin and others of dual origin ( 4 , 5 and for a review, see 6 ).
This specific technique can be completed by the use of cocultures in vitro, in
which one of the tissue compartments can be modified (overexpression or
inhibition of basement membrane components or of regulatory molecules) to
analyze the consequences on the basement membrane composition and on the
resulting extracellular matrix-cell signaling (7 , 8) . Interspecies or heterotopic
(from different levels of the gastrointestinal tract) recombinants have also been
used successfully to approach the regulation of the expression of functional
markers, i.e., digestive enzymes (9-11) .

2. Materials
2.1. Tissue Dissection
2.1.1. Dissection of the Embryos

1. Paraffin support: Mix melted paraffin (Histomed standard, Labo Moderne, Paris)

with activated charcoal (Merck, Darmstadt, Mannheim, Germany). Pour the
mixture into small glass dishes. After cooling, cover them with aluminium foil.
Sterilize at 110 C for 1 h. Let cool at room temperature. These supports provide

accentuated contrast for the dissection of transparent embryos.

°

2. 9% NaCl solution: 9 g NaCl made up to 1 L. Sterilize by autoclaving.

2.1.2. Preparation of Gelified Medium
75% Ham's F-10 medium (Gibco, Life Technologies, Gaithersburg, MD),
25% agar solution at 1 g/100 mL, and 2 mg/mL gentamicin (Septigen 40,
Schering Plough, Kenilworth, NJ).

1. Hank's solution: 137 m M NaCl, 5 m M KCl, 2 m M CaCl 2 , 1 m M MgCl 2 , 0.8 m M

Na 2 H PO 4 , 0.22 m M KH 2 PO 4 , 0.28 m M MgSO 4 , 5 m M glucose.