WORKSHOP BOOK Fluorescent in Situ Hybridisation - FISH - on Human Sperm

Post Hybridization Wash
The aim of this stage is to remove all the non-specific hybridized sites that
might have been created dur<strong>in</strong>g the hybridization.
Remember to keep your samples <strong>in</strong> dark dur<strong>in</strong>g the whole procedure, as light
will reduce the fluorescence of the probes
Firstly, place your samples <strong>in</strong> 2X SSC for some seconds, just to remove the
coverslips easily.
Adjust the temperature of SSC solution <strong>in</strong> the jar.
Put your slides <strong>in</strong> jars with SSC (for chromosome 18 wash <strong>in</strong> 0.25M SSC and for
chromosomes 13, 21, X and Y <strong>in</strong> 0.4M SSC). The jars should be <strong>in</strong>cubated <strong>in</strong>
water-bath at 74 o C for 2 m<strong>in</strong>utes exactly.
After the bath, wash with Water For Injection or Phosphate Buffered for 1
m<strong>in</strong>ute, <strong>in</strong> order to remove the rema<strong>in</strong><strong>in</strong>g SSC without agitation.
F<strong>in</strong>ally, samples are dried <strong>in</strong> ethanol (gradually 70%, 90% and 100%) for 2
m<strong>in</strong>utes <strong>in</strong> each dilution.
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Sta<strong>in</strong><strong>in</strong>g
At this stage, spermatozoa are sta<strong>in</strong>ed, to be visualized under the microscope.
For sta<strong>in</strong><strong>in</strong>g, 3-10μl of DAPI Antifade are added <strong>in</strong> each marked region and
covered with glass coverslip.
It is important to discrete the marg<strong>in</strong>s of each cell, so as to be sure of the
sign that you get.
Coverslips are placed carefully above each marked region and are secured <strong>in</strong>
place with a little polish.
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