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

<strong>FISH</strong> on Spermatozoa
SOP
Preparation
6. Procedure
6.1 Preparation
Wash your sample, dilut<strong>in</strong>g 1ml with 4ml PBS and centrifuge for 7m<strong>in</strong> at 2000 rpm.
Remove the supernatant and resuspend the pellet. Dilute it with 1ml fresh PBS and centrifuge
for 7m<strong>in</strong> at 2000 rpm.
Repeat the second wash for one more time.
TIP: Resuspend your pellet before add<strong>in</strong>g the fresh PBS.
Dur<strong>in</strong>g the last centrification, prepare your fixative solution. Fixative should be cool and
fresh.
FIXATIVE SOLUTION
Prepare FRESH fixation solution 3:1 Methanol/Acetic Acid
For f<strong>in</strong>al volume of 4 ml mix
• 3ml of methanol
• 1ml of CH 3COOH
At the end of the 3 rd wash remove all the supernatant and resuspend the pellet.
Add your fixative drop-to-drop, to dilute your sample.
Leave the sample on the fridge (4 o C) for 1 hour.
After 1 hour, remove the sample from the fridge and centrifuge for 3m<strong>in</strong>.
Remove all the supernatant and resuspend the pellet.
Add fresh fixative drop-to-drop.
Clean your slides at Ethanol and pour your sample drop-to-drop to have a good spread<strong>in</strong>g.
Hands-on Workshops, Thessaloniki, 30.08 – 5.09 2014 2