Nectin-2wt/wt and nectin-2LacZ/LacZ mouse testes were fixed for 4 h in 4% paraformaldehyde before freezing in OCT cryoprotectant (Tissue-Tek, Sak-ura Finetek, Torrance, CA). Cryosections (thickness, 10 |xm) were collected on poly-L-lysine-coated slides, air-dried, and postfixed in cold 1:1 methanol: acetone for 90 min at —20°C. Sections were blocked with PBS containing 5% normal horse serum and 2% normal goat serum (PBS/HG), followed by overnight incubation at 4°C with primary antibodies in PBS/HG. The following primary antibodies were used: anti-nectin-2 rat monoclonal antibodies (mAbs) 17B10 and 6B3, anti-nectin-3 rat mAb 103-A1 (1:5 diluted hybridoma supernatants for each), anti-espin mouse mAb 31 (2.5 |xg/ ml; BD Transduction Laboratories, San Jose, CA), and a rabbit polyclonal antibody against p-galactosidase (1:500; Molecular Probes, Eugene, OR). After 1 h of washing with PBS, the sections were incubated with combinations of either Cy3-conjugated anti-rat (1:1000; Jackson Immuno Research, West Grove, PA) and Alexa488-conjugated anti-rabbit antibodies (1: 500; Molecular Probes) or Cy2-conjugated anti-rat (1:200; Jackson Immuno Research) and Cy3-conjugated anti-mouse antibodies (1:1000; Jackson Immuno Research). Nuclei were counterstained by addition of 100 ng/ml of Hoechst 33258 (Molecular Probes). The sections were washed extensively with PBS and mounted with Immu-Mount (Shandon, Pittsburgh, PA). Images were acquired on a Zeiss Axioplan II fluorescence microscope equipped with a model SP401 camera (Diagnostic Instruments, Inc., Sterling Heights, MI) and processed with Adobe Photoshop software.