Immunofluorescence Staining of Mouse Testes

Frozen sections of mouse testes were prepared at 5 μM thickness, permeabilized with 0.1% Triton X-100 in PBS for 20 min, and boiled for antigen retrieval in 20 mM citrate buffer (pH 6.0) for 10 min [27 (link)]. After blocking with 3% goat serum in PBST overnight at 4°C, the sections were then incubated with a 1:2000 dilution of anti-EFCAB2 antiserum as the primary antibody for 1 h, followed by incubation for an additional hour with a 1:1000 dilution of the secondary antibody Alexa Fluor 594 (Life Technologies, Gaithersburg, MD, USA) together with PNA-lectin, a FITC-conjugated peanut agglutinin (Sigma; L7381). The sections were counterstained using DAPI and observed under a fluorescence microscope (BX50/BX-FLA; Olympus, Tokyo, Japan).