Unless otherwise stated, all reagents were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO). Enhanced chemi-luminescence Western blotting detection kit was purchased from Amer-sham (Arlington Heights, IL). Polyclonal antiprohibitin antibody and preimmune rabbit serum were purchased from Neomarkers (Fremont, CA), monoclonal antiproliferating cell nuclear antigen (anti-PCNA) antibody from Santa Cruz Biotechnology (Santa Cruz, CA), and polyclonal anticholesterol side-chain cleavage cytochrome P450 (P450scc) was from Chemicon International, Inc. (Temecula, CA). Goat anti-mouse immunoglobulin (Ig) G (H+L) conjugated to Alexa Fluor 488, goat anti-rabbit IgG conjugated to Alexa Fluor 594, and 4′,6′-diamidino-2-phenylindole were purchased from Molecular Probes (Eugene, OR).
Animal and Cell Preparations
Female Sprague-Dawley rats (age, 23 days) were separated into three groups (n = 15 rats/group). In the first group, animals were injected s.c. with saline, followed 24 h later by normal (preimmune) rabbit serum (NRS; 0.1 ml). In the second group, animals were injected s.c. with 15 IU of eCG, followed 24 h later by NRS (0.1 ml). In the third group, animals were injected s.c. with 15 IU eCG, followed 24 h later by anti-eCG (0.1 ml). In all groups, animals were killed with an overdose of pentobarbital 24 h after the last hormone/antibody injection.