Assay of Total Luteal Protein
Specific sets of corpora lutea obtained in experiment 3 were homogenized in 500 ^l modified RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA) containing detergents (1% Nonidet P-40 and 0.015% sodium deoxycholate) using a tissue homogenizer. The homogenate was diluted (1:30) for assay of total protein using the Bio-Rad Protein Microassay. All standards and samples were assayed in triplicate.
Quantitation of Macrophages and Apoptotic Nuclei
Numbers of macrophages per high-power field were determined for coded slides by visual observation of immu-nodetectable cells. A light microscope with a X45 objective was used. For experiment 1, one section was examined to determine macrophage numbers, and as many fields as possible were counted for each corpus luteum in the section (4-23 corpora lutea). New ovulations on estrus were easily distinguishable in whole ovarian sections and were not included as corpora lutea for this analysis. For experiment 2, two sections of the frozen group of corpora lutea (processed in different staining runs) were examined in the same manner as for experiment 1 (1-3 fields in 3-7 corpora lutea), and an average number of macrophages per high-power field was obtained for each rat. proventil inhaler
For quantitation of apoptotic nuclei, two sections from each ovary or group of corpora lutea were examined as above, using coded slides, and an average number of apop-totic nuclei per high-power field was obtained. Again, as many fields as possible were counted for each corpus lu-teum in these sections, and new ovulations were excluded from analysis of whole ovarian sections.
Comparisons among multiple groups were carried out using the Bonferroni multiple comparisons test. Comparisons between two groups were carried out using Student’s ?-test.