9 Human Sperm Cells
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- Dapi-solution: per slide, mix 1 ml 2 x SSC/0.1 % NP-40 with 1 III Dapi Counterstaining
stock solution (plus 1111 propidium iodide stock solution if a red fluorochrome is not being used to detect DNA probe)
- Dapi stock solution: dissolve 10 mg DAPI (Serra) in 10 ml aqua bidest
and store aliquots of 50 III at -20°C (the aliquot in use can be stored at
4°C)
- Propidium iodide stock solution: dissolve 10 mg propidium iodide
(Sigma) in 10 ml aqua bidest and store aliquots of 50 III at -20°C
(the aliquot in use can be stored at 4°C)
- Antifade: Mounting medium for fluorescence (Vectashield)
Procedure
Preparation of sperm cells
1. Suspend ejaculate in 8 ml PBS.
2. Centrifuge at 1250 rpm (280 g) for 10 min.
3. Discard supernatant and resuspend in 8 ml PBS.
4. Centrifuge at 1250 rpm (280 g) for 10 min.
5. Discard supernatant and resuspend in 8 ml PBS.
6. Centrifuge at 1250 rpm (280 g) for 10 min.
7. Discard supernatant.
8. While vortexing semen sediment add 20 drops of Otto's solution drop
by drop, then suspend in 8 ml Otto's solution and incubate at 37°C for
30 min.
9. Centrifuge at 1250 rpm (280 g) for 10 min.
10. Discard supernatant.
11. While vortexing pellet add 20 drops ice-cold fIxative drop by drop, then
add 8 ml fIxative.
12. Centrifuge at 1250 rpm (280 g) for 10 min.
13. If there is a little pellet, drop it onto a slide immediately, if there is a
very visible pellet, wash 3 times with 4 ml fixative and then drop onto a
slide.