Hi Everyone
I try to make some time lapse for sperm meiosis process. I followed the standard protocol to dissect 2-3 males in 3.2 ul SM Buffer in the slide and then put a 18x18
cover slip on top of that and the side of the cover slip is sealed to control evaporation. Then time lapse movies are made by using spinning disc confocal. I got a few movies.
The main problem in this process is to find out enough primary spermatocytes. In some cases, I waited for a few minutes to see whether primary spermatocytes start Metaphase-I but by that time media is dried out.
I will be grateful to have some advice to make those movies. I am particularly looking for:
- which stage of male I should use to get more primary spermatocytes, so that I can capture the entire meiosis process?
- I tried to dissect worms on coverslip instead of slide. But that didn’t work well. So I am not sure which other ways I can try?
- I tried with 2% agarose pad which is used for making in utero time lapse of fertilization process. That was worst !!!
- How long I can wait for a primary spermatocyte to see whether it starts Metaphase-I ?
Thanks 