Mayer s haematoxylin solution

For H&E staining of the millimetre-thick bovine muscle tissue, the bovine muscle tissue was fixed with 4% PFA, dipped in 30% sucrose solution at 4 °C, and subsequently frozen in optimal cutting temperature compound (Sakura Finetek Japan Co. Ltd., Tokyo, Japan). The frozen tissue was cut with a cryostat chamber (Hyrax C25, Carl Zeiss Co. Ltd.) as 10-μm-thick sections; the sections were mounted on glass slides and stained with Mayer’s haematoxylin solution (Wako Pure Chemical Industries Ltd.) and 0.5% eosin Y ethanol solution (Wako Pure Chemical Industries Ltd.). We observed the H&E-stained sections under a microscope (M80, Leica Microsystems Ltd., Wetzlar, Germany).

Testes and epididymides were fixed overnight at 4°C in Bouin’s fluid (16045–1, Polysciences, Inc., Warrington, PA, USA) and embedded in paraffin. Paraffin sections (5 μm) were rehydrated, treated with 1% periodic acid for 20 min at room temperature, and incubated with Schiff's reagent (193–08445, FUJIFILM Wako Pure Chemical, Osaka, Japan) for 20 min at room temperature. The sections were stained with Mayer's haematoxylin solution (131–09665, FUJIFILM Wako Pure Chemical) and observed using a BX-50 microscope (Olympus, Tokyo, Japan).
Cauda epididymal spermatozoa were dispersed in TYH medium [50 ] or non-capacitating medium [51 ] for 10 min before the observation of morphology and motility. For mitochondria staining, spermatozoa were dispersed in TYH medium containing 10 nM MitoTracker Red FM (M22425, Thermo Fisher Scientific) and 10 μg/mL of Hoechst 33342 (H3570, Thermo Fisher Scientific). After 10 min incubation at 37°C under 5% CO₂ in air, spermatozoa were observed using a phase contrast microscope (BX50, Olympus).