Fisher superfrost plus microscope slides

Mice were killed by inhalation of CO₂, and the epididymides were harvested and fixed by immersion in periodate-lysine-paraformaldehyde containing 4% paraformaldehyde for ∼5 h at room temperature as described previously [20 (link), 37 (link)] and rinsed three times in PBS for 5 min each. Tissues were cryoprotected by incubating them in a solution of 30% sucrose in PBS for at least 24 h. Tissues were embedded in OCT compound (Tissue-Tek; Sakura Finetek), mounted on a cutting block, and frozen. The tissue was then cut at 10- to 16-μm thickness using a Leica 3050 cryostat (Leica Microsystems). Sections were picked up onto Fisher Superfrost /Plus microscope slides (Fisher Scientific) and refrigerated until use. Sections were hydrated in PBS and heated by microwaving in an alkaline buffer (Vector Laboratory) three times for 2 min each time, with 5-min intervals for antigen retrieval. To block nonspecific-binding, 1% bovine serum albumin in PBS was applied for 30 min at room temperature. The sections were then incubated with primary antibodies in a moist chamber for 90 min at room temperature or overnight at 4°C. The samples were washed in PBS and incubated with secondary antibodies for 60 min at room temperature. All antibodies were diluted in DAKO antibody diluent (DAKO).