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Heart Rate
7 eggs were collected and incubated for 6 days. Using
forceps the embryos were removed from the eggs and placed in
warm Howard's ringers solution. With fine forceps, the trunk
was severed above and below the heart region. The dorsal
region of the embryo was removed and the heart left intact.
The heart was transferred to fresh warm Howard's ringers
solution. 1.0 mg of nicotine was added directly to 4 of the
hearts. Heart rate was measured by counting the beats every
15 seconds and then allowing 15 seconds to pass before the
next reading. The procedure for the 3 controls was the same,
except they were treated with 1.0 mg of saline solution
instead of nicotine.
Staining
for Limb Cartilage
2, 13 day old nicotine treated embryos and 3, 13 day old
control embryos were isolated for staining. Legs and wings
were fixed in 5% TCA for 1 hour. The limbs were then washed
3 times with PBS and immersed in Alcian green stain for
approximately 18 hours. They were washed 3 times with 70%
ethanol, and once with 85% ethanol, 95% ethanol, and 100%
ethanol. Limbs were then washed 3 times with methyl
salicylate and left in the solution for 4 days. Staining was
observed and images were captured using an Olympus DP10
camera.
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