SEM analysis of testis

Testes (from mice at age of 9–18 weeks) dissected from mice were cut into 5-mm-thick slices with a safety razor, fixed for 1 h at 4 °C in 0.1 M phosphate buffer (pH 7.4) containing 1% OsO₄, immersed in 50% dimethyl sulfoxide, and then crushed with the use of a TF-2 apparatus (Eiko). The samples were washed with phosphate buffer, transferred to 0.1% OsO₄ in phosphate buffer, and incubated for 48 to 72 h at 20 °C. They were then fixed again for 1 h at 4 °C with 1% OsO₄ in phosphate buffer, stained for 2 h with 2% tannic acid (Fujifilm Wako Pure Chemical) and for 1 h with phosphate buffer containing 1% OsO₄, dehydrated with a graded series of ethanol solutions (50, 70, 90, 100, 100, and 100%), and subjected to critical point drying with the use of a Samdri-PVT-3D system (Tousimis). The specimens were finally mounted on sample stages, coated with osmium with the use of an HPC-30W device (Vacuum Device), and observed with an S-4800 field-emission scanning electron microscope (Hitachi).