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