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

Issue Date:
05/12/2012
Version:
1
Rev.:
0
SOP-LAB-01: <strong>FISH</strong> ON HUMAN SPERM
Page #:
6 out of 7
Prepared by: Glykeria Samolada Edited by: Vassilis Katsares Approved by: Alexia Chatziparasidou
6.3 Co-denaduration
Firstly, add 3-10µl of your probe <strong>in</strong> each marked region and cover with glass coverlsip (small round or 20X20). Avoid
expos<strong>in</strong>g your samples on light, to keep the levels of fluorescence for sample’s count<strong>in</strong>g.
Before the hybridisation, samples should be co-denaturated to obta<strong>in</strong> s<strong>in</strong>gle-stranded DNA both at probes and at the
sperm DNA. For co-denaturation, slides are placed on a hot plate at 74 o C. (For chromosome 18 leave the slides at 74 o C
for 2 m<strong>in</strong>utes and for the chromosomes X, Y, 13 and 21 for 5 m<strong>in</strong>utes, or accord<strong>in</strong>g to probe’s supplier guides).
6.4 <strong>Hybridisation</strong>
For the hybridization, <strong>in</strong>cubate the samples overnight <strong>in</strong> 37 o C, <strong>in</strong>side a humid and dark box.
6.5 Post-<strong>Hybridisation</strong> 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.
For the post-hybridisation wash, 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.
6.6 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.
6.7 Scor<strong>in</strong>g & Count<strong>in</strong>g
Fields should be found at 10X or 20X magnification. Then, magnification should be <strong>in</strong>creased to 40X and gradually to
100X with oil, to count the spots.
There should be only one spot of each chromosome <strong>in</strong> every sperm cell. (Chromosomes 18, X and 13at our experiment
emit green colour, whereas chromosomes Y and 21 emit red colour).
TIP:
• Spot count<strong>in</strong>g should be careful. The marg<strong>in</strong>s of the cells should be clear <strong>in</strong> order to know that the spots come
from chromosomes.
• 2 spots <strong>in</strong> a cell should be clearly separated and have the same <strong>in</strong>tensity <strong>in</strong> order to be considered as aneuploid.
(Blanco et al, 1996)
SOP-LAB-02: <strong>FISH</strong> ON HUMAN SPERM/01/00/05-12-2012