WORKSHOP BOOK Fluorescent in Situ Hybridisation - FISH - on Human Sperm
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5. Exam<str<strong>on</strong>g>in</str<strong>on</strong>g>e slides<br />
Microscopy - Bulbs<br />
• 100 Watt mercury bulb recommended<br />
• Do not use bulbs over their recommended life<br />
• Do not use tungsten bulbs<br />
• Ensure bulb is correctly centred/focused<br />
• x10 and x60 objectives m<str<strong>on</strong>g>in</str<strong>on</strong>g>imum<br />
• Phase c<strong>on</strong>trast essential for assess<str<strong>on</strong>g>in</str<strong>on</strong>g>g samples<br />
• Use s<str<strong>on</strong>g>in</str<strong>on</strong>g>gle filters for clear image / count<str<strong>on</strong>g>in</str<strong>on</strong>g>g<br />
• Filters last at most 3 - 4 years!<br />
29<br />
6. Slide Scor<str<strong>on</strong>g>in</str<strong>on</strong>g>g<br />
Scor<str<strong>on</strong>g>in</str<strong>on</strong>g>g criteria<br />
1. overlapped spermatozoa or sperm heads without a<br />
well-def<str<strong>on</strong>g>in</str<strong>on</strong>g>ed boundary are not counted<br />
2. <str<strong>on</strong>g>in</str<strong>on</strong>g> cases of disomy or diploidy, all signals should<br />
have the same <str<strong>on</strong>g>in</str<strong>on</strong>g>tensity and be separated from each<br />
other by a distance l<strong>on</strong>ger than the diameter of each<br />
signal<br />
3. nullisomies are not directly scored and are<br />
c<strong>on</strong>servatively c<strong>on</strong>sidered as equivalent to the<br />
<str<strong>on</strong>g>in</str<strong>on</strong>g>cidence of disomies<br />
Blanco J. et al (1996) Hum Reprod 11: 722–726<br />
30
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