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Experimental Procedure Preparation of Nicotine Solution and Treatment of Chick Embryos Prepare nicotine solution by adding 25 µL of nicotine stock solution (40% weight / volume) to 10 mL of Howard’s Ringer’s Solution. Make a small opening in the blunt end of the eggs, and administer 0.1 ml of nicotine solution to 8 of the eggs, 0.2 mL of the nicotine solution to 12 of the eggs, and 0.1 mL of Howard’s Ringer’s Solution to 2 of the remaining eggs and 0.2 mL of Howard’s Ringer’s Solution to the other two eggs as the controls. Incubate eggs at 37 °C for 14 days. After two weeks, remove the now 17-day-old chicks from the eggs and continue with the staining protocol. Protocol for Staining Specimen for Bone and Cartilage Fix specimen in 4% paraformaldehyde for at least 48 hours at low temperature. Rinse embryos for at least 1 day in distilled water, with several changes. Postfix in 70% ethanol. Embryos may be stored indefinitely. Remove feathers and skin, and eviscerate specimens. Place specimen in solution of Alcian Blue cartilage stain (20 mg Alcian Blue for every 100 mL of 70% ethanol in glacial acetic acid) for 2–3 days. Destain specimen by transferring it to 70% ethanol in glacial acetic acid for 1 hour, then to 100% ethanol for 1 day. Change frequently. Soak specimen in distilled water for 1 day. Transfer embryo to 2% KOH overnight to clear embryo. Transfer to 2% KOH to which enough saturated Alizarin Red has been added to turn the solution a very dark purple color. Transfer to plain 2% KOH to rinse out Alizarin Red. The solution should be changed once or twice over a 24 hour period. When cartilagenous skeletal structures, stained blue, and bone, stained red, become visible, transfer specimen to 1% KOH / 20% glycerol for one day to allow clearing of the tissue, occasionally gently swirling. Store specimen in 50% ethanol / 50% glycerol in a screw top jar. Photograph embryos and compare development of bone and cartilage.