Plasma testosterone levels were measured using an ELISA adapted from an earlier radioimmunoassay method as detailed elsewhere. The limit of detection was ~12 pg/ml.
The PLP-fixed epididymides of control and treated 25-day-old rats were cryoprotected by immersion in 30% sucrose in PBS for at least 4 h. The entire epididymis (head, body, and tail) was then mounted in OTC compound Tissue-Tek (Miles Inc., Elkhart, IN) and frozen at —29°C in a Reichert Frigocut cryostat (Reichert Jung, Derry, NH). Sections were cut at 4 pm, picked up onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA), and kept at 4°C.
For immunostaining, the sections were hydrated for 10 min in PBS, and antigen retrieval was performed by incubating the slides for 4 min with 1% SDS in PBS. After three washes in PBS for 5 min each, nonspecific staining was blocked by applying to the slides a solution of 1% BSA in PBS/0.02% sodium azide for 15 min. An affinity-purified rabbit primary antibody against the 56-kDa subunit of the vacuolar H+-ATPase was applied to the slides at a concentration of 1:1000 in DAKO diluent (DAKO Corporation, Carpinteria, CA) for 90 min at room temperature. Sections were then washed twice for 5 min each time in PBS containing 2.7% NaCl to reduce nonspecific binding, followed by one wash in normal PBS. For immunofluorescence labeling, a secondary goat anti-rabbit antibody coupled to the fluorophore CY3 (Jackson Immunologicals, West Grove, PA) was applied to the slides at a 1:800 dilution in DAKO diluent for 1 h at room temperature, followed by the same series of washes that were used for the primary antibody. The slides were mounted in Vectash-ield diluted 1:1 with Tris buffer pH 8.5 (Vector Laboratories, Burlingame, CA).