I used Eosin Y for analyzing viability of mouse sperms that had good movement in pre-stained analysis, but after staining, none of them was colorless, and almost all of them had been stained similarly (smooth pink). Is it normal? In my other investigations, the live sperms were colorless and the dead ones were pink. What do you think?
You may have already considered this, but all live-dead ("viability") stains will eventually stain all of the cells, both living and dead. You must determine the optimal incubation time for the vital dye with the cells. In the case of sperm this is facilitated by the fact that you know the motile cells are alive. So you want to find an incubation time that does not result in staining the motile sperm. The optimal staining time is different for each species and is sensitive to other incubation conditions such as temperature and composition of the medium. Good luck!
Perhaps, you have to find an optimal concentration and incubation period and avoid the cells dying during processing.
@Mukesh. thank you for your answer. I dissected caudal epididymis too free the sperms and incubated for 30-40 minutes. After that, I observed them without staining and they were perfect. Then, instantly, I stained them and during this very short period they whole got blue color. I doubt that they are dead altogether and that fast.
The vital staining technique sperm we generally use Eosin Y and Nigrosin staining method, which is a standard method. This is a water based dye prepared and matured and strained before use. If you use alcohol based. all sperm will be stained whether the are having intact or damaged membrane. This stain is now not considered as Live and dead cell differentiation. This represent the plasma membrane intact ness of sperm at the time of staining of sperm and represent vitality of the sperm. So we now call the percentage of eosinophil and non-eosinophilic sperm i.e intact plasma membrane or damaged plasma membrane. A sperm may be live and motile, however if the membrane is damaged it will take stain. Duration of exposure and making the smear and drying is also important in sperm evaluation with this stain. The epididymal sperm are different to this stain ; in comparison to ejaculated sperm. This is used for evaluation of ejaculated sperm ; which is fully matured and mixed with seminal plasms.
Do consider all these aspect before using these stain for evaluation of sperm. At a higher magnification we can differentiate stained and non stained sperm better. At this maginfication it is confusing.
The answer of dr. Mohanty is very instructive. I only underline that without nigrosin counterstaining, the evaluation is difficult. Pavel Travnik
@ dear dr Mohanty and Travnik: thank you so much for your kind answer. It was so comprehensive and useful.
For me, not movement is not similar to dead, mostly the time you can have not motility sample, but in live state.
I think you must do it a control for you staning solution, used fresh sample, and kill anohter for observed the diferent coloration, in deep the liught of your microscope can do it a wrong observation and of course used other system for check you protocol, for example phenol red or other vital staning. and the most important, count the cells, for present numbre of cells in the different condition.
Dear Mr. Mansouri,
for these useful comments and suggestions I can add the protocol for prepearing Eosin and Nigrosin, what we use in our laboratory. Eosin stock solution: 0,5 g E + 100 ml physiological saline (buffered with Sörensen buffer), Nigrosin solution: 1 g + 100 ml destilled water. We use this staining for bull semen, sampels are processed as follows; 1 drop of sample (watch glass) + 1 drop of Eosin (20s mixing) + 2 drops of Nigrosin - smear it and let it dry.
@Ondrej: Thank you so much, I really appreciate. It was great. Thnx
I used negorsin in PFA 10%, for keep the simple more time if you want to leave a few day the simple before you take images.
Hi Ardalan! Actually, I am having the same problem. I need to analyse mouse sperm viability and I have tried the protocols I used to use for human sperm (eosin Y alone or eosin-nigrosin) but none is working well. I was wondering if you have already optimized any of these protocols for mice. And in affirmative case, could you share it with me? Thanks in advance.
Hi Ardalan
I'm also working on viability staining of mouse sperm (eosin-nigrosin) and have experienced a high percentage of dead sperm after staining (70-80%) with my current protocol. If you have an optimized protocol, would you share it with me? Thanks in advance!
Hello All!
I realise this is now quite an old thread, but I have been experiencing the same problems as mentioned here in mouse sperm (high proportion stained) and, like many of you, wondered if someone could please provide me with an optimised/suggested protocol? I'd be very grateful!
I also wondered, once you've made your eosin and nigrosin stocks up (in PBS and distilled water, respectively) how do you store them? Do you keep them at RT? And how long do you find they are good for?
Many thanks and best wishes,
Emma