Control incubations were performed using antibodies that had been preabsorbed with an excess of the immunizing peptide (11 amino acids that correspond to the C-terminus of the 56 kDa, B1 subunit of the H+-ATPase) prior to the first incubation step.
Quantification of H+-ATPase Immunoexpression
Study 1. Black-and-white photographs were taken of the whole epididymis of each animal. The number of H+-ATPase positive cells on each photograph was counted and the basement membrane of the associated tubule was drawn around using a Wacom graphics tablet (Vancouver, WA). The mean number of immunopositive cells per millimeter of epididymal tubular basal membrane was calculated for each animal.
Study 2. Sections were examined using a Nikon Eclipse 800 epifluores-cence microscope (Micro Video Instruments Inc., Avon, MA). Black-and-white images were obtained using a Hamamatsu Orca CCD digital camera (Micro Video Instruments Inc.) and IP lab Spectrum software (Scanalytics, Vienna, VA). The images were taken using a 20X objective.
Cells with positive fluorescence staining for vacuolar H+-ATPase in the epididymides were counted on digital images while they appeared on the computer screen. The basal perimeter of the epididymal tubules that were analyzed was measured using IP lab Spectrum software. The final number of positive cells per millimeter in the perimeter of the basal membrane of the tubule was calculated for each image in Microsoft Excel. The number of rats analyzed in each cohort is listed in Table 1.