Meiotic chromosome surface spreads

Preparation of Mouse Fetal Ovary spread for cytological analysis

1. Collection of Embryonic Ovaries

C57BL/6J female mice were mated with male mice overnight. The following morning, females were examined for the presence of vaginal plugs. The day on which a vaginal plug was detected was designated embryonic day (E) 0.5. At E17.5, pregnant females were euthanized in accordance with the protocol approved by the relevant Ethics Committee. The uterus was removed, and the embryos were dissected and transferred to ice-cold phosphate-buffered saline (PBS). The embryos were decapitated, and the ovaries were carefully dissected under a stereomicroscope. The ovaries were transferred to fresh, ice-cold PBS. The surrounding mesonephric tissue was carefully removed using fine forceps.

2. Preparation of Ovarian Cell Spreads. Each ovary was transferred into a 30 µL drop of 100 mM sucrose. Under a stereomicroscope, the ovary was gently fragmented into several small pieces using fine forceps. Using a pipette fitted with a 20 µL tip, the tissue fragments were gently pipetted up and down approximately 30 times, directing the liquid against the bottom of the dish to facilitate cell release. Pipetting was performed gently to minimize mechanical damage to the cells and potential chromosome breakage. Once a cloudy cell suspension had formed, 15 µL of the suspension was transferred onto each of two clean glass slides.

3. Fixation and Slide Preparation. To each slide containing 15 µL of cell suspension, 100 µL of freshly prepared 1% paraformaldehyde (PFA) containing 0.1% (v/v) Triton X-100 was added. The slides were placed in a humidified chamber at room temperature (RT) and incubated for 2–4 hours. Following incubation, the slides were removed from the humidified chamber and allowed to air-dry under the chemical hood. The dried slides were washed four times for 1 minute each in distilled water containing 0.4% (v/v) Kodak Photo-Flo solution. The slides were then air-dried completely and stored at −80 °C until further use.

4. Preparation of Paraformaldehyde Solution

Preparation of an 18.5% (w/v) PFA Stock Solution. Fresh 18.5% (w/v) PFA solution can be prepared from paraformaldehyde powder using the following procedure. Appropriate chemical safety precautions must be followed throughout preparation. Add 4.8 mL of distilled water to a 50 mL conical tube suitable for the intended heating procedure. Add 0.925 g of paraformaldehyde powder. Add 35 µL of 1 N KOH. Tightly cap the tube and place it in a glass beaker containing approximately 200 mL of water. Heat the beaker in a microwave until the surrounding water begins to boil. Remove the beaker and gently vortex the tube to facilitate dissolution of the paraformaldehyde. If necessary, filter the solution to avoid insoluble particles. Repeat the heating and mixing steps until the paraformaldehyde has dissolved completely. Allow the solution to cool on ice.