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50 (median tissue culture infectious dose) per milliliter of virus. The inoculum was instilled atraumatically into the vaginal vault. Another 1-ml dose was similarly introduced 4 hours later. To prevent loss of the inoculum, between and after inoculations the tranquilized animals were propped up in their cages so that the inoculum would drain toward the cervix. Four animals each 1,3, and 7 days later, and 2 animals 12 days later, were euthanized. Tissues were collected and fixed in 10% neutral buffered formalin or Molecular Biology Fixative (Streck Laboratories, Omaha, NE). Animal maintenance and procedures were in accordance with the guidelines of the Committee on Animals for the Harvard Medical School and the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, DC, 1996).
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1 (25 units/ml) in 1× RWS. After washing in RWS, 2× SSC, and 0.1× SSC, sections were dehydrated in graded ethanot solutions containing 0.3 M ammonium acetate and then dried and coated with Kodak NTB-2 emulsion exposed at 4°C, developed, and stained.
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2 and diaminobenzidine according to the manufacturer's protocol (ABC Elite kit, Vector 2 Labs, Burlingame, CA). After washing in PBS and 0.1× PBS, sections were dehydrated in a series of graded ethanol that contained 0.3 ammonium acetate and coated with nuclear track emulsion. After development the slides were briefly counterstained in hematoxylin.
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Supported by the Great Lakes Regional Center for AIDS Research; NIH grants AI 28246, AI 38565, and RR 00168; and the German Ministry of Education and Research (BMBF 01 K1-97146). We thank L. Shand, C. O'Neill, T. Leonard, and G. Sedgewick for preparation of the manuscript and figures and M. Dykhuizen and R. Evans for technical assistance.
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