Dapi anti fade mountant

Slides in which sperm chromatin decondensation was performed were fixed with 10% formaldehyde for 12 min, washed with PBS and dehydrated in an ethanol series (70%, 90%, and 100%) for 2 min each. Afterwards, DNA was denatured through incubation in 0.5 M NaOH for 4 min, again dehydrated in ethanol series and dried horizontally. Next, 15 µL of the Peptide Nucleic Acid (PNA) probe (TTAGGGTTAGGGTT, TelG, Panagene; Korea) at a final concentration of 0.8 µg/mL in hybridization buffer (10 mM Na₂HPO₄, 10 mM NaCl, 20 mM Tris, 70% formamide, and pH = 7.4) was denatured at 72 °C for 6 min and subsequently poured on the top of each sample. Following this, slides were covered with parafilm and placed for hybridization in a small, damp container at room temperature for 1 h in the dark.
After hybridization, parafilm was carefully removed and three washings were conducted: first, in PBS + 0.1% Tween20 at room temperature and darkness for 2 min; second, in the same solution at 48 °C and darkness for 20 min; and, finally, with 2× SSC + 0.1% Tween 20 at room temperature and darkness for 2 min. Slides were subsequently dehydrated in an ethanol series (70%, 90%, and 100%), dried horizontally, and counterstained with DAPI Antifade Mountant (Thermo Fisher Scientific; Waltham, MA, USA). Slides were stored at 4 °C until microscope images were captured, which was performed on the following day.