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

Tip for spread<strong>in</strong>g
• Keep your slide humid
• Keep your sample quite high and leave it to fall on the slide
Observe the slides carefully under the microscope to decide how much time each slide it will
need for treatment.
NaOH helps to remove any debris from your sample and also de-condense the spermatozoa.
Samples with condensed spermatozoa heads or a lot of debris need to be treated with NaOH
for a longer period of time (2-8 m<strong>in</strong>).
TIP: Cryopreserved samples are usually more compact than fresh samples, so they need a
longer treatment with NaOH at this stage.
Hands-on Workshops, Thessaloniki, 30.08 – 5.09 2014 3
Treatment
At this stage, samples are treated with NaOH 0.5M. Each slide is treated for the period of
time that was decided previously (under the microscope).
After treatment with NaOH, samples are washed twice <strong>in</strong> fresh SSC 2X (SSC consists of NaCl
and Sodium Citrate), <strong>in</strong> order to remove the rema<strong>in</strong><strong>in</strong>g NaOH from your samples. Each wash
lasts for 5 m<strong>in</strong>utes
F<strong>in</strong>ally, samples are dried <strong>in</strong> ethanol (gradually 70%, 90% and 100%) for 5 m<strong>in</strong>utes <strong>in</strong> each
dilution.
Observe the slides carefully under the microscope and decide where the probe is go<strong>in</strong>g to be
added. Mark the selected region, at which your sample is better treated
Hands-on Workshops, Thessaloniki, 30.08 – 5.09 2014 4