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However, the cytosolic staining for Cl seemed stronger compared to that of C2. Double labeling for the E2 subunit (Fig. 5, B, B, E, and E ) revealed colocalization of both C isoforms with E2 in subapical vesicles (Fig. 5, C, C0, F, and F0; yellow staining). In contrast, Cl and C2 were barely detected in apical microvilli (Fig. 5, C, C0, F, and F0; red staining). No significant staining was detected in principal cells. Localization of the G Subunit of the V-ATPase: G1 and G3 Isoforms Similarly to the A and C subunits, strong apical and weaker cytosolic staining for both Gl and G3 were detected in narrow cells (data not shown), and clear cells of the epididymis (Fig. 6, A and B) and vas deferens (data not shown). Double labeling for the E2 subunit (Fig. 6, C and D) showed colocalization of Gl and G3 with E2 in clear cells (Fig. 6, E and F). No significant staining was seen in principal cells. Interestingly, epididymal sperm showed immunoreactivity for the G3 isoform (Fig. 6B) but not for the Gl isoform (Fig. 6A). The localization of the brain-specific G2 isoform was not examined. FIG. 5. Localization of the V-ATPase C1 and C2 subunit isoforms. Rat cauda epididymidis was stained with antibodies against subunit isoforms C1 (A and A0; green) and C2 (D and D0; green) and were double-stained for E2 (B, B0, E, and E0; red). C and C0 and F and F0 show merged images for C1 and E2 and for C2 and E2, respectively. Both C1 and C2 are located in clear cells, where they colocalize with E2 in subapical vesicles (yellow staining in C and F). No significant C1 and C2 staining in E2-labeled apical microvilli was detected (red staining in C0 and F0). Weaker intracellular staining was also detected in the cytoplasm of clear cells, with a stronger staining for C1 compared to C2. No staining was detected in principal cells. Bar = 15 im (A-F) and 5 im (A0-F0). FIG. 6. Localization of the G1 and G3 isoforms of the V-ATPase. Rat cauda epididymidis was stained with antibodies against subunit isoforms G1 (A; green) and G3 (B; green) and were double-stained for E2 (C and D; red). Both G1 and G3 are located in the apical pole of clear cells. The merged images shown in E and F show colocalization of G1 (E; yellow-orange staining) and G3 (F; yellow-orange staining) with E2 in subapical vesicles, with no apparent colocalization in E2-labeled microvilli (red staining in E and F). No staining is seen in principal cells. Epididymal sperm are stained for G3 but not for G1. Bar = 15 im.