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Specificity of the V-ATPase Antibodies Each of the V-ATPase subunit antibodies used in this study, except the novel anti-subunit A antibody, has been characterized previously. The specificity of these antibodies in epididymis samples was further confirmed by Western blotting. As shown in Figure 1, all antibodies gave one single band at the appropriate molecular weight, showing their purity. A more complete analysis was performed for the novel anti-A antibody by repeating the Western blot after preincubation of the antibody with the immunizing peptide. This antibody revealed a single band at the expected molecular weight of 70 kDa on Western blots from epididymis homogenates (Fig. 1, lane A), andnosignalwas detected after preincubation of the antibody with the A subunit peptide. Localization of the A Subunit of the V-ATPase Immunofluorescence using our affinity-purified anti-subunit A antibody revealed a strong apical staining in the apical pole of narrow cells of the initial segments (Fig. 2A). Double labeling for the A (Fig. 2B) and E2 (Fig. 2c) subunits revealed their colocalization in narrow cells (yellow staining in Fig. 2D). A strong apical staining was observed for subunit A in clear cells of all regions of the epididymis, including the cauda (Fig. 3A), as well as the vas deferens (data not shown). TABLE 1. Revised nomeclature of Human V-ATPase subunit isoforms (based on Smith et al. ). FIG. 1 Detection of V-ATPase subunits by Western blot in total homogenate from rat epididymis. All antibodies used in the present study showed one single band at their appropriate molecular weight (A, 70 kDa;C? and C2, 42 kDa;G? and G3, 13 kDa;a?, a2, a4, 116 kDa;d? and d2, 38 kDa). The signal was completely abolished after preincubation of the anti-subunit A antibody with the immunizing peptide, showing specificity of this novel antibody. FIG. 2 Localization of the V-ATPase A subunit in narrow cells. A 5-im section of rat initial segments was stained with antibodies against the A and E2 subunits of the V-ATPase. Subunit A (A; green) is located in the apical pole of a subpopulation of epithelial cells. Double labeling for the A (B; green) and E2 (C; red) subunits showed that these subunits colocalize in the apical pole of narrow cells (C; yellow). Bar = 15 im (A) and 10 im (B-D). FIG. 3. Localization of the V-ATPase A subunit in clear cells. A 5-im rat cauda epididymidis section was stained with antibodies against the A and E2 subunits of the V-ATPase. Subunit A (A; green) is located in the apical pole of clear cells identified by their E2 expression (B; red). The merged image of C shows colocalization of A and E2 in apical microvilli and subapical endo-somes (C and inset;yellow staining). A faint staining was also detected in the cytosol of clear cells. No staining was detected in principal cells in this distal portion of the epididymis. The arrows indicate the border between apical microvilli and subapical vesicles. D) Immunogold electron microscope localization of subunit A in a clear cell from the cauda epididymidis. Subunit A is located in apical microvilli as well as in the subapical pole of the cell. Bar = 50 im (A-C), 5 im (insets in A-C), and 500 nm (D).