Dear Veerle

I found halosperm step by step (for DNA fragmentation evaluation), please do it.

STEP BY STEP

1. Heat the eppendorf tubes in a microwave or in a water bath mantained at 100ºC for 5

minutes, to melt the agarose.

2. Transfer the tubes to a water bath mantained at 37ºC for 5 minutes.

3. Add 25 μL of the semen sample and mix.

4. Place 15 μL of the mixture on the slide and cover with a coverslip.

5. Put the slide on a cold surface and introduce it in the fridge for 5 minutes the agarose to solidfy.

6. Add 80 μL of the denaturing solution to 10 ml of distilled water.

7. Take the slide from the fridge and remove the coverslip.

8. Incubate the slide in the denaturing solution for 7 minutes.

9. Remove the slide from the denaturing solution and incubate in the lysis solution for 25

minutes.

10. Remove the slide from the lysis solution and incubate in distilled water for 5 minutes.

11. Incubate the slide in 70% ethanol for 2 minutes.

12. Incubate the slide in 90% ethanol for 2 minutes.

13. Incubate the slide in 100% ethanol for 2 minutes.

14. Leave to dry at room temperature.

15. Stain with Diffquick.

best regards

vahid

@ Vahid Esmaeili Borzabadi:

Dear Sir, that's exactly what it did. I did follow the protocol you described above step by step (the only thing that differs is the staining but we also use the one suggested by HaloTech). Sometimes it works and sometimes it doesn't, even within the same experiment (that is when I use the same sperm sample and the products from the same kit (duplo analyses)). That's what's so weird about the whole thing... Did you ever experience some problems with HaloSperm? What sperm samples did you use? Were the samples fresh or thawed? Thank you in advance! Kind regards, Veerle

@ Alireza Mohammadi:

Dear Sir, thank you for your reply! When I observed the rippled membrane of the sperm cells, I also thought we were dealing with an osmolality problem. However, since we only use products provided within the kit, this should not be the case... Also, we see a difference in results between the slide that were in the same incubation tray, even though sperm samples were the same. Kind regards, Veerle

Hello, Veerle.

I work with ther original version of SCD test (Fernández y col., 2003) and I stain with a Wright solution composed by Wright, glycerol, acetone and H2Odd and, always, always it work... This solution must be fresh on each occasion, the excess must be discarded. Any question that you have, feel free to ask! =)

I will work with halosperm kit this week, I replay to you about that.

Dear Yoeli,

We also performed an experiment with the original version just to see whether there were some problems with the new kit and we everything worked just fine! However, we already bought 10 kit and since its a lot of money, we don't want to throw it away. What exactly is the composition (concentrations?) of your staining solution? Thank you in advance!

Kind regards,

Veerle

Dear Vahid,

Thank you for your efforts!

Kind regards,

Veerle

Stock Solution:

Glycerol 5ml

Metanol 5ml

Wright 0.1gr

Working Solution

Stock Solution 4ml

Acetone 3ml

PBS 2ml

H2Odd 31ml

For the Stock Solution you need mix glycerol and wright in a mortar to homogenize the stock, then add metanol. Next step is make the working solution and use it. You can immerse your slide in a petri dish with the working solution by 5-10 minutes occasionally blowing, then wash gently with fresh water.

The stock solution can be storage by few months, nevertheless the working solution need be make for each day.

I hope it can serve.. Kind regards!

Hello,

I encountered the same problem when I used the Halomax Kit for bull 2 years ago.I followed the protocol as it was presented, step by step. I stained the slides with Wright Stain but no halos were observed. Samples from the same bulls were also tested with SCSA. I intend to use the test again this year but I am trying to find more info regarding the problem I encountered back then. Did you find any solutions to the problem you encountered?

Hi Andra,

The pH of the PBS buffer seemed to be crucial. It should be exactly 6.88.

In other occasions, the slides just need a second (longer) staining step.

Hope this helps,

Kind regards

Thank you very much for sharing your experience. I shall try with PBS, P.H 6.88 and see if it works and maybe a second staining step.

Best Wishes.

If you are using commercially available Halosperm kit, make sure that you adhere to the storage conditions recommended by the manufacturer. I noticed similar problem after leaving buffers at the bench overnight (it should be refrigerated after use).