WORKSHOP BOOK Fluorescent in Situ Hybridisation - FISH - on Human Sperm
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Co-denaturati<strong>on</strong><br />
Firstly, add 3- 10µl of your probe <str<strong>on</strong>g>in</str<strong>on</strong>g> each marked regi<strong>on</strong> and cover with glass coverlsip (small<br />
round or 20X20). Avoid expos<str<strong>on</strong>g>in</str<strong>on</strong>g>g your samples <strong>on</strong> light, to keep the levels of fluorescence for<br />
sample’s count<str<strong>on</strong>g>in</str<strong>on</strong>g>g.<br />
Before the hybridizati<strong>on</strong>, samples should be co-denaturated to obta<str<strong>on</strong>g>in</str<strong>on</strong>g> s<str<strong>on</strong>g>in</str<strong>on</strong>g>lge-stranded DNA<br />
both at probes and at the sperm DNA. For co-denaturati<strong>on</strong>, slides are placed <str<strong>on</strong>g>in</str<strong>on</strong>g> a hot plate<br />
at 74 o C. (For chromosome 18 leave the slides at 74 o C for 2 m<str<strong>on</strong>g>in</str<strong>on</strong>g>utes and for the chromosomes<br />
X, Y, 13 and 21 for 5 m<str<strong>on</strong>g>in</str<strong>on</strong>g>utes, or accord<str<strong>on</strong>g>in</str<strong>on</strong>g>g to probe’s supplier guides) .<br />
Hands-<strong>on</strong> Workshops, Thessal<strong>on</strong>iki, 30.08 – 5.09 2014 5<br />
Hybridizati<strong>on</strong><br />
For the hybridisati<strong>on</strong>, <str<strong>on</strong>g>in</str<strong>on</strong>g>cubate the samples overnight <str<strong>on</strong>g>in</str<strong>on</strong>g>37 o C,<br />
<str<strong>on</strong>g>in</str<strong>on</strong>g>side a humid and dark box.<br />
Hands-<strong>on</strong> Workshops, Thessal<strong>on</strong>iki, 30.08 – 5.09 2014 6
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