9 Human Sperm Cells
133
3. While slide is air drying, denature specific DNA probe in 10 III hybridization mix for 5 min at 75°C and place on ice immediately.
4. Place denatured probe (1O Ill) on one part of the slide, cover carefully
with a 20x20 mm coverslip avoiding air bubbles and seal with rubber
cement.
5. Incubate in a moist chamber at 37°C over night or at least 4 h for centromeric probes.
Posthybridization washes
For commercially available probes, follow the instructions of the manufacturers or:
1. Carefully remove rubber cement and coverslip with forceps.
2. Place slide in a Coplin jar with I x SSC in a 75°C water bath for 2 min,
agitate the slide for the first 1-3 s.
3. Wash slide in 2x SSC/O.I % NP-40 at room temperature for 5 s-I min.
Counterstaining
1. Place I ml DAPIIpropidium iodide working solution on each wet slide
and incubate for 5 min in the dark.
2. Place slide shortly in 2x SSC/O.I % NP-40 and air dry slide in the dark.
3. Apply 20 III of antifade in a row of dots across the slide and cover with a
coverslip.
4. Store slide at -20°C in dark.
Results
Analyze slides with a suitable filter set with a fluorescence microscope. Be
aware that normal sperms are haploid (Fig. 3)! According to several studies, most autosomes have a disomy frequency in sperms of approximately I per 1000. Therefore depending on the study purpose, usually
about 10,000 sperms per donor have to be analyzed. To distinguish disomy
Storage
133
3. While slide is air drying, denature specific DNA probe in 10 III hybridization mix for 5 min at 75°C and place on ice immediately.
4. Place denatured probe (1O Ill) on one part of the slide, cover carefully
with a 20x20 mm coverslip avoiding air bubbles and seal with rubber
cement.
5. Incubate in a moist chamber at 37°C over night or at least 4 h for centromeric probes.
Posthybridization washes
For commercially available probes, follow the instructions of the manufacturers or:
1. Carefully remove rubber cement and coverslip with forceps.
2. Place slide in a Coplin jar with I x SSC in a 75°C water bath for 2 min,
agitate the slide for the first 1-3 s.
3. Wash slide in 2x SSC/O.I % NP-40 at room temperature for 5 s-I min.
Counterstaining
1. Place I ml DAPIIpropidium iodide working solution on each wet slide
and incubate for 5 min in the dark.
2. Place slide shortly in 2x SSC/O.I % NP-40 and air dry slide in the dark.
3. Apply 20 III of antifade in a row of dots across the slide and cover with a
coverslip.
4. Store slide at -20°C in dark.
Results
Analyze slides with a suitable filter set with a fluorescence microscope. Be
aware that normal sperms are haploid (Fig. 3)! According to several studies, most autosomes have a disomy frequency in sperms of approximately I per 1000. Therefore depending on the study purpose, usually
about 10,000 sperms per donor have to be analyzed. To distinguish disomy
Storage