European Medical Alliance

Preparation of Sperm Slide

1. Freshly ejaculated sperm sample is allowed to liquefy at room temperature for 30 min.

2. Dilute 1 mL of sperm in 1X PBS (1:10) and centrifuge for 5 min at 500,.

3. Remove the supernatant and resuspend the pellet in 10 mL of 1X PBS for a new wash by centrifugation (5 min at 500, ).

4. Resuspend the pellet in 1 mL of fresh fixative (3:1 methanol:glacial acetic acid).

6. Using a Pasteur pipet, drop one droplet of the sperm suspension on a clean microscope slide from a height of approx 10 cm.

7. The slide is air-dried and checked under the light microscope (x10 and x40) to ensure that both the cell concentration and the spreading are optimum (see Note 2). Using a diamond marker, draw a circle on the underside of the slide to mark where the spermatozoa are.

8. Store the slide for 2 d at room temperature, preferentially in a hermetic box in order to avoid dust deposits (see Note 3).

9. Immediately before the PRINS reaction, the slide is denatured in 0.5 MNaOH at room temperature for 4 min (see Note 4).

10. Wash the slide twice for 2 min at room temperature in 2X SSC.

11. Pass the slide through an ethanol series (70%, 90%, and 100%), 2 min each step, and air-dry.