1 2 3 4 5

		Procedure: 1.Cut small pieces of Whatmann 3MM filter paper (3mm X 3mm) and soak them with 3 ml Hydrocortison-acetate (3 mg/ml in ethanol) (Sigma). Air-dry the filter discs for 1.5 hrs. 2.Obtain 4mL each: bFGF (1.5 mg/ml), VEGF (1.5 mg/ml), or DMEM (a tissue culture medium in which the control filter discs should be soaked). Pour the solutions into separate petri dishes that are labeled with the names of the compounds. Soak the filter paper disks in the solutions for 30 minutes. 3.While the filter paper is soaking, obtain ten-day-old chick eggs and wash them in 70% ethanol. Label the eggs as either DMEM, bFGF, or VEGF using a pencil. Open the blunt end of the egg with forceps. Peel back the shell membrane, being careful not to damage the CAM membrane.     4.Drop the filter paper over the embryo in the area with the least number of visible blood vessels. Cover the hole in the shells with Scotch tape, and place in an incubator set at 37C (being careful not to jostle the embryos) for four days. 5.After the four-day period, remove the tape covering the holes and extract the filter paper using a pair of forceps. Examine the filter paper for the presence of blood vessels. Count all blood vessels on the filter paper and record the numbers within your lab notebook. Compare to the other conditions. Instructor's prep sheet Lab Protocol