Cm1950 frozen microtome

Fresh testis was cut into fragments about 5 mm³ and fixed with 4% paraformaldehyde at 4 °C overnight, dehydrated and rehydrated with gradient methanol, and then immersed in 30% (w/v) sucrose at 4 °C overnight. The fragments were embedded in the Tissue-Tek OCT compound (SAKURA Tissue-Tek, Atlanta, GA, USA), and then sectioned at 4 µm with a Leica CM1950 frozen microtome (Leica). Probes were synthesized according to the operation guide of DIG RNA Labeling Mix (Roche). The sample slides were hybridized with 1 µg/mL DIG probes in seal boxes at 65 °C overnight. Following hybridization, the slides were washed with washing buffer (Roche) and blocked with Blocking Reagent (Roche) for at least 1 h. Probe signals were developed with an AP-conjugated anti-DIG antibody (Roche; diluted 1:2000) and colored with NBT/BCIP Stock Solution (Roche). Photographs were imaged by a microscope (Leica). Probe primers are listed in Table 1.

The 12 samples were cut into blocks (0.5 ± 0.1 mm³). The blocks were secured to the tray with frozen section embedding agent (Leica, Germany), which then placed in a Leica CM 1950 frozen microtome (Leica, Germany) to cool at a low temperature. Then, the secured blocks were sliced into slices of 50 μm along transverse. The slices was soaked in chloral hydrate transparent solution (Merck, USA) for a few minutes and laid flat on the slide. The structure of all slides was observed under a Nikon 80i light microscope (Nikon, Japan).