Tissue Recombinants to Study Extracellular Matrix Targeting to Basement Membranes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 301

end of the egg and then drop the chorioallantoic membrane by aspirating about
5 mL of albumen with a sterile syringe. Cover the opening with sterile scotch
tape and incubate the eggs at 38 ° C for an additional 24 h in the same position ( see

Notes 3 and 4 ).

2. Grafting procedure:

• Cut an opening of approx 1-cm diameter in the shell above the chorioallantoic

membrane using forceps and curved scissors.

• To gain access to the embryo, open tactfully the vitellin and the amniotic

membranes with the needle-mounted probe and with the help of forceps.

• Make a sharp incision in the coelom of the chick embryos near the intersection

of the major blood vessels and then carefully and gently implant the graft.

• Seal the opening in the shell with sterile scotch tape and reincubate the eggs

at 38 C in the humidified incubator up to the wished developmental stage of

the explants.

°

3. The grafts are recovered at various times after implantation.

• Decapitate the chick embryo after its removal out of the egg.

• Fasten the embryo laterally with pins on both sides; make an incision longitu-

dinally along the middle of the embryo.

• Locate the grafts with the aid of the carbon particles; the implants which have

developed their own vascularization, can be found anywhere within the lat-
eral coelom, the visceral loops or underneath the lungs. Recover the grafts
and handle for subsequent analysis.

3.3. Immunodetection of Basement Membrane Molecules
on Interspecies Intestines
1. Preparation of the sections ( Fig. 2 )

•

Prepare a cork support of about 1 cm 2 ; deposit on it a drop of Tissue-Tek
compound (O.C.T. Compound, Miles Inc., USA)

• Embed the hybrid intestine vertically in the Tissue-Tek compound and imme-

diately freeze in isopentane (Prolabo) prealably cooled in liquid nitrogen.
These inclusions can be kept for several years at -40 ° C.

• Cut transverse sections of about 5-6- C using a cryostat and

place the sections on SuperFrost/Plus Microscope slides (Menzel-Gläser,
Germany). Store at -20

µ m thick at -25 °

° C until use.

2. Immunofluorescence

• Prepare the required amount of antibody dilutions in NaN 3 -containing PBS.

These aliquots can be kept up to 1-2 mo at 4 C

• Add onto each section, an aliquot of first antibodies and incubate the sections
for 1 to 2 h at room temperature in a humidified chamber.
•Wash the sections several times with PBS.
•

°

Add the corresponding secondary trimethyl-rhodamine or fluorescein
isothiocyanate (TRITC or FITC)- labeled antibodies at the optimal dilution
indicated by the manufacturer. Place the humidified chamber in dark or cover
it with an aluminium foil.