Isolated left testes obtained from EAO-affected mice and control mice were placed in OCT compound (Miles Laboratories, IL, USA), frozen in liquid nitrogen, and stored at −80 °C until use. Five-micrometer-thick sections were cut with a cryostat (CM1900; Leica, Wetzlar, Germany) and then fixed in ethanol for 10 min at −20 °C. The sections were then rinsed in phosphate-buffered saline (PBS) and incubated with Block Ace (Yukijirushi, Hokkaido, Japan) for 20 min at room temperature to inactivate endogenous peroxidase activity. After rinsing in PBS, the sections were incubated with a rat anti-mouse B220 (clone: RA3-6B2, ×200; BD Biosciences) monoclonal antibody, followed by incubation with rabbit anti-rat IgG (Vector Labs, CA, USA) at room temperature. Bound antibodies were detected by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ZyMax, South San Francisco, CA) at room temperature. Immunoreactive cells were visualized using a Vectastain ABC Kit (Vector Labs, CA, USA) with 3,3′-diaminobenzidine (DAB) as the chromogen. Bound HRP was detected using 0.05% DAB and 0.01% H2O2. Sections processed with rabbit serum instead of the primary antibodies were used as negative controls. The stained sections were counterstained with methylgreen (Vector Laboratories, CA, USA).