Description
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Procedures - Freezing
- Pick up the cauda epididymis from male C57BL/6J mouse, remove adherent adipose tissue and blood, and put two cauda epididymis into 100 µL of R18S3 on a petri dish.
- Fix the cauda epididymis with forceps and cut the ductus epididymis about ten times. After that, shake the petri dish for 1 min and suspend medium to make sperm equal.
- Connect the straw with 1 mL syringe. Sealing protocol is as follows.
- Aspirate 100 µL of HTF (used as a weight) and prepare 1 cm airspace at the edge of straw.
- Use above syringe and aspirate 10 µL of sperm-suspension prepared at step (2). And then prepare 1 cm airspace at the edge of straw.
- Seal the edge of straw with heat sealer.
- Detach the straw from the syringe and seal another edge of straw.
4. Put the sealed straw into float, with turning sperm-suspension layer to the bottom.Put the straw-fl oat on the surface of liquid nitrogen in N2 tank.
Keep it stand for 15 min.
5. After 15 min, soak it into liquid nitrogen.
Thawing
- Pick up the freezing straw from liquid nitrogen, and soak it into warm water at 37 ℃ .
Keep it stand for 15 min. - After 15 min, pick up the straw from warm water and wipe it with some papers.
After that, cut the both edges of straw and drop the sperm-suspension on the petri dish. - Pick up 2 µL of sperm-suspension and apply it into 200 µL HTF, which is pre-warmed in incubator
(37 ℃ , 5% CO2 in air) beforehand.
After 1 hour from above treatment, observe the motility and active level.
