In Vivo Angiogenic Activity of Erythropoietin - Tissue-Protective Cytokines: Methods and Protocols - page 222

Here, we have used the CAM assay to demonstrate the angio-
genic activity in vivo of rHuEpo.

2 Materials

2.1 Reagents

1. White fertile chicken eggs obtained at day 1-2 postlaying (see
Note 1).
2. 70% ethanol in H 2 O.
3. Sterile routine tissue culture medium: minimum essential
medium (MEM) with nonessential amino acids.
4. Sterilized gelatin sponges (Gelfoam, Upjohn Company,
Kalamazoo, MI, USA).
5. Bouin's solution.
6. Toluidine blue 0. Use a 0.5% aqueous solution.

2.2 Equipment

1. Egg incubator (Kemps Koops—Eugene, Oregon, USA).
2. 25-26 gauge hypodermic needles and 1 ml syringes.
3. Pasteur pipets.
4. Curved-tip forceps.
5. Straight-tip forceps.
6. Small dissecting scissors.
7. Stereomicroscope (SXX9, Olympus Italia).
8. Light microscope (BX51, Olympus Italia).

3 Methods

1. Sterilize all instruments in 70% ethanol prior to use.
2. Clean the fertilized white chick eggs with 70% ethanol and
incubate at 37°C and 60% humidity in an egg incubator for
48 h (see Note 2).
3. Aspirate 2-3 ml albumen from the egg using 25-26 gauge
hypodermic needle and 1 ml syringe at the pin hole of the egg
on day 3 of incubation. This procedure creates a false air sac
directly over the CAM, allowing its dissociation from the egg
shell membrane (see Note 3).
4. After albumen removal, cut a square window into the shell
with the aid of scissors. This window can be enlarged to
approximately 10 × 10 mm. The underlying embryo and CAM
vessels are revealed. Seal the windows with transparent tape or
a glass coverslip of the same dimension, and return the egg to
the incubator. This step should be done in an enclosed area