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Localization of the a Subunit of the V-ATPase: a1, a2, and a4 Isoforms Isoforms al and a4 were detected in the apical pole of narrow cells (data not shown) and in clear cells of the epididymis (Fig. 7, A and B) and vas deferens (data not shown). Double labeling for the E2 subunit (Fig. 7C) revealed that al colocalized with E2 in subapical vesicles but was absent from the microvilli (arrows in Fig. 7, A, C, and E). Subunit a4 showed a complete colocalization with E2 in subapical vesicles and microvilli of clear cells (Fig. 7, B, D, and F). Expression of the osteoclast-specific a3 isoform was not examined. The distribution of the a2 isoform was quite distinct from that of the al and a4 isoforms. Whereas no significant staining was detected in the apical pole of narrow and clear cells (data not shown), subunit a2 was detected in intracellular structures of both clear and principal cells, with a weaker staining detected in clear cells. The staining was much stronger in the proximal regions of the epididymis, including caput, corpus, and proximal cauda (Fig. 8A), and was not detectable in the distal cauda (data not shown). Similarly to the A subunit, double labeling for TGN38 revealed that a2 is present in structures that are closely (but not exactly) associated with the TGN38-stained trans-Golgi network (Fig. 8, B-D). FIG. 7. Immunolocalization of the V-ATPase a1 and a4 subunit isoforms. Rat cauda epididymidis was stained with antibodies against subunits a1 (A; green), a4 (B; green), and E2 (C and D; red). a1 and a4 are located in the apical pole of clear cells but show different patterns of staining. a1 colocalizes with E2 in subapical vesicles (E; yellow) but is absent from E2-labeled microvilli (E; red;arrows). A bright staining was observed for a4, which colocalizes with E2 in both subapical vesicles (F; yellow) and E2-labeled microvilli (F; yellow-orange staining;arrows). Bar = 15 im. FIG. 8. Intracellular localization of the V-ATPase a2 subunit isoform in the proximal cauda epididymidis. A) Conventional immunofluorescence localization of a2 (A; green) in intracellular structures of principal cells. Nuclei are stained with DAPI (blue). B, C, and D) Confocal microscopy showing a proximal cauda epididymidis section stained for a2 (B; green) and TGN38 (C; red). a2-labeled intracellular structures are closely associated but are not identical to the TGN38-labeled trans-Golgi network (D; merged panel;red and green staining). Bar = 5 im.