THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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ù'lhen the anirnals were killed, bloods were collected_and
centrifuged after one hour in the cord room to obtain the serum:
ïhe serum samples were immediately frozen at -20 c untiì use. At the
same time, the entire luternized ovaries were removed, trimmed, and inrnedlately
frozen at -20 C unti'l further analysis.
(iJ) Progesterone assay-
serum progesterone was quantitated by radioirmunoassay u*ììzing
antisera developed by Merck Institute, Rahway , New ,jersey, USA (a gïft from
Dr. D. Grinwich). The assays u,ere conducted according to the methoc described
by orczk et al (1974) with slight modifications. The procedures were
descríbed as fol I ows:
a- Extracti on:
0.'l ml of rat serum was extracted with 5 vol of petroleum
ether(Fisher, anarytical grade). The extracts were drÍed under nitrogen. Then,
appropriate volume of 0.0rM p's containing 0.0r% gelatin were added.
In order to determine the percentage of progesterone that
has been extracted, 10,000 cpm of 3H-progesterone was added lnto each
sample of sera, and extracted as described above to determine the efflciency
of extractÍon
b- Assay:
To each assay tube was added 0..l ml of pBS containing varlous
concentratfons of progesterone or 0.1 ml of unknown samp'le wfth appropriate
di'lution, 0..l ml of 3H-prooesterone (.15-20,000 cprn), and 0.ì ml of antibodles