Ab200839

Epididymal sections (5 μm thick) of adult male WT and V1a receptor–deficient mice were prepared in the same way as for the in situ hybridization analysis. The slides were incubated in incubation buffer (1% fetal bovine serum and 0.3% Triton X-100 in PBS) containing anti-V-ATPase B1 and B2 subunit antibodies (ab200839, 1:1,000; Abcam) overnight at RT. After washing with 0.3% Triton X-100–containing PBS, tissue slices were incubated in the incubation buffer containing the secondary antibody (Alexa Fluor 488–conjugated anti-rabbit IgG [A-11034], 1:200; Thermo Fisher Scientific) for 1 h at RT. DAPI (Dojindo) was used as the nuclear marker. Sections were then mounted using ProLong Gold antifade reagent (Thermo Fisher Scientific), and images were obtained under a fluorescent microscope (AX80). To demonstrate the specificity of the primary antibody, experiments were performed in which epididymal sections were incubated with incubation buffer alone instead of the primary antibody as a negative control.
For double staining of the V1a receptor mRNA and V-ATPase protein, in situ hybridization analysis was performed following the above-mentioned protocol. Upon detecting the signals of the Avpr1a mRNA, immunohistochemistry staining for the V-ATPase protein was performed. All immunohistochemistry experiments were repeated on tissues from three different male mice, with the same results.

To investigate the spatial distribution of active Cre recombinase, tdTomato signals in various tissues from 5-month-old Lcn9-Cre; Rosa26^tdTomato mice were examined with a fluorescence stereomicroscope (M165; Leica). To determine changes over time, tdTomato signals in Lcn9-Cre; Rosa26^tdTomato mice of different ages were detected.
To visualize the precise distribution of active Cre recombinase and track the cell types with positive signals, epididymal samples isolated from the heterozygous Lcn9-Cre; Rosa26^tdTomato males were embedded in Tissue-Tek OCT (4583; Sakura). Six-μm thick frozen sections were washed with phosphate-buffered saline (PBS), blocked with 10% goat serum and incubated with primary antibodies overnight at 4°C. Immunofluorescence signals were screened after FITC-conjugated secondary antibody incubation and counterstaining with DAPI. Primary antibodies included cytokeratin 5 (Krt5) (ab52635; rabbit; Abcam; 1:200) [36 (link)] for basal cells, α-SMA (BM0002; mouse; Boster-bio; 1:200) for smooth muscle cells, and vacuolar H⁺-ATPase (V-ATPase) (ab200839; rabbit; Abcam; 1:200) [37 (link)] for narrow cells and clear cells. FITC-conjugated goat anti-mouse IgG (ZF-0312; ZSGB-bio; 1:100) and goat anti-rabbit IgG (ZF-0311; ZSGB-bio; 1:100) were used as secondary antibodies. Fluorescent images were captured with a confocal microscope (LSM900; ZEISS).