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 #:
5 out of 7
Prepared by: Glykeria Samolada Edited by: Vassilis Katsares Approved by: Alexia Chatziparasidou
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 CH3COOH
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 <strong>in</strong> the fridge (4 o C) for 1 hour.
After 1 hour, remove the sample from the fridge and centrifuge for 3m<strong>in</strong> at 2000 rpm.
Remove all the supernatant and resuspend the pellet.
Add fresh fixative drop-to-drop.
Clean your slides with Ethanol and pour your sample drop-to-drop to have a good spread<strong>in</strong>g.
TIPS FOR SPREADING:
• 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 (0.5M) 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.
6.2 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.
SOP-LAB-02: <strong>FISH</strong> ON HUMAN SPERM/01/00/05-12-2012