Hello,
please does anybody work with Yo-Pro 1/ PI protocol for staining apoptotic and necrotic sperms in stallion ejaculate?
I have tried to use same protocol as for bovine sperms but it doesn't work well. The protocol was, briefly - two times washing with FCS (1%) Saline,1500 rpm/7 min. Concentrations of Yo - Pro 1 and PI were 5 micromoles, 5 micrograms/ml, respectively. Incubation of 25 microliters of washed sample with staining solution was proceed in ambient temperature for 20 minutes. After the 4 microliters of sample and 4 microliters of DAPI/Vectashield were mounted on a slide and counted under fluorescent microscope.
In case of stallion sperms, signal is very low I was not be able to recognize the categories.
Please, does anybody have any suggestion about concentrations of fluorescent stains?
Thank you.
Ondřej
Hi Ondrej,
I have actually used the PI/Yopro-1 protocol for equine sperm in flow cytometry protocols. I attach you the link to the paper. We usually used 1.167 uM PI and 75 nM Yopro (final concentration), and incubated it for 15 minutes at 37 degrees. I'd suggest to increase your incubation temperature and length if you fail to see appropiate fluorescence. On the other hand, you could try to look at it fresh (avoiding the vectashield). I have sometimes had some troubles when using antifade for non-fixed sperm. I attach you the link to the protocol in case you find it useful. good luck!
Bea
http://www.ncbi.nlm.nih.gov/pubmed/21436310
Dear Beatriz,
muito obrigado - Thank you very much for your answer it is very helpful for our team.
My best regards,O
Ondrej
I hope this helps, but feel free to ask anything you need! good luck!
Bea
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