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Ms. Miertschin asked on behalf of Houston's Thomas Street Clinic if there was a chance of having the new application available in Spanish. Mr. Haught responded that once the new application was finalized, it will indeed be professionally translated into Spanish. The next meeting was scheduled for January 7, 2005, and is tentatively at the Texas Animal Health Commission, from 12-3 pm. Dr. Keiser announced a break from 12: 45 to 1: while the MAC awaited the arrival of Ms. Melchior with a budgetary update. Upon returning from break, it was announced that Ms. Melchior would apparently be unable to attend the meeting, so the budget report would instead be sent to the MAC members via e- mail. The motion to adjourn was unanimously approved at 1: 07.
Animals. Male Sprague-Dawley rats weighing between 250 and 350 g were used in all experiments. The aorta, caudal, femoral, iliac, mesenteric resistance, renal and superior mesenteric arteries were removed and processed according to the experimental protocols described below. Immunocytochemistry. After removal, the blood vessels were cleaned of extraneous tissue and cryoprotected by immersion in increasing amounts of sucrose in PBS 15% for 2 days, 30% for 2 days ; at 4C. The vessels were then placed in O.C.T. mounting media and quick-frozen 50C in isopentane cooled with dry ice 13- m sections were cut on a cryostat. Sections were placed on poly-Llysine-coated, circularly etched slides and stored at 80C until use. Sections were thawed, fixed in cold 4% paraformaldehyde for 10 min and then washed. These sections were incubated in blocking solution PBS containing 10% normal goat serum, 1% bovine serum albumin and 0.1% Triton X-100 ; for 2 hr at room temperature and then incubated with alpha-1B AR antisera 1: 100150 dilution ; for 24 hr at the blocking solution without goat serum. Slides were then washed in PBS followed by application of an affinity-purified FITCconjugated goat anti-rabbit IgG 1: 200 dilution; Jackson Immunologicals, West Grove, PA ; secondary antibody. After 1 hr at room temperature, the secondary antibody was removed by washing with PBS, and the slides were coverslipped with Vectashield Vector Laboratories, Burlingame, CA ; mounting media and then examined by fluorescence microscopy. The anti-receptor IgG, originally described by Fonseca et al. 1995 ; , was affinity-purified against the immunizing peptide immobilized on a solid support matrix Sulfolink; Pierce Chemicals, Rockford, IL ; . Preimmune IgG was prepared by fractionating rabbit serum over the same column. Laser scanning confocal microscopic analysis of immunofluorescent images. Microscopic sections were placed on the stage of an inverted microscope and imaged using a laser-scanning confocal microscope RCM 8000; Nikon, Melville, NY ; . FITC fluorescence was excited using the 488-nm wavelength output of an argon laser. Emitted fluorescence was collected using a Nikon 40 water-immersion objective N.A. 1.15 ; . Image quality was enhanced by averaging 64 scans image. Images were immediately stored on an optical disk recorder as eight-bit, 512 483 pixel TIFF files. The files were later transferred to an 80486 microcomputer for off-line analysis using the program Metamorph Universal Imaging, West Chester, PA ; . Fluorescence measurements were confined to the vascular smooth muscle regions between the highly autofluorescent elastin bands. This was facilitated by setting the analysis program threshold settings to exclude the bright elastin autofluorescence. The mean fluorescence in these defined regions was calculated for both preimmune and postimmune arterial sections. Differences in these mean fluorescent values were determined by a t test for unpaired data. In vitro assessment of contractile function. Isolated blood vessel segments were prepared by techniques routinely used in our laboratory Piascik et al., 1994, 1995 ; . Arterial segments were removed and placed in a cold PSS of the following composition in mM ; : NaCl, 130; KCl, 4.7; KH2PO4, 1.18; MgSO4 7H2O, 1.17; CaCl2 2H2O and enoxaparin.

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