Nb300 229

The testes were freshly dissected from adult mice (7 wk) and decapsulated. The testes were ground in PBS, and the cells were collected and centrifuged. The cell pellets were washed several times in PBS and suspended in hypotonic buffer (30 mM Tris at pH 7.5, 17 mM Tris sodium citrate, 5 mM EDTA, and 50 mM sucrose). After keeping for 5 min, the cells were centrifuged and the supernatant discarded, followed by making suspension in 100 mM sucrose. The cell suspension was spread onto slides dipped in formaldehyde solution (1% formaldehyde, 0.15% Triton X-100 in water adjusted with sodium borate to pH 9.2). Slides were then placed in a humidified chamber overnight and air-dried at room temperature. For immunostaining, slides were rinsed for 5 min in PBS, and then incubated for 30 min in blocking buffer (3% BSA, 10% normal goat serum in PBS). The slides were incubated with primary antibodies for Scp1 (NB300-229, Novus) and Scp3 (sc-74569, Santa Cruz Biotechnology) in blocking buffer overnight at 4°C, followed by incubation with goat anti-mouse IgG (H + L) Alexa Fluor 568 (A11004) and goat anti-rabbit IgG (H + L) Alexa Fluor 488 (A11008) (Invitrogen) secondary antibodies and DAPI for 1 h at room temperature. The slides were then washed with PBS and mounted.