Solutions

See Suppliers for the requisite materials.

Zero Calcium Seawater:
See zero calcium seawater in Modified Seawaters.

Pre-Isolation Mix:
To make 100ml of mix, you must mix fresh daily:

To make 100 ml of 2M MgCl2 from the 4.9M stock take 41 ml of the 4.9M stock and dilute to 100ml with distilled water.

To make KEGTA, 0.2M, take 7.6g of EGTA and add to 80 ml of distilled water. While stirring add one pellet at a time, potassium hydroxide. It will take approximately 25-30 pellets to bring the pH up to 6.6. This takes awhile! Be patient. It is best to use a pH meter, but paper will do. When it gets closer, as evidenced by the fact that most of the EGTA is dissolved, you may want to switch to a KOH solution instead of the pellets. A 2N solution should work fine. Finally bring the volume up to 100ml. This solution will keep at room temperature in a stoppered bottle for several months.

Isolation Mix:

You will need a chemical called Triton X100 (see Suppliers).

Make up the Pre-Isolation Mix and remove 10 ml. To this add 0.1 ml of Triton X100. This will be a little hard to dissolve. Be patient. Gentle rocking seems to work best.

Procedure

To embryos at or near metaphase: (or any stage you wish to view)

1. Hand centrifuge embryos and re-suspend in zero calcium seawater two times.
2. Hand centrifuge embryos and re-suspend in PERM MIX two times.
3. Hand centrifuge embryos and add small amount of ISOLATION MIX.
4. Let sit for 30 minutes.
5. View under phase microscope.

← live vs. isolated ↑

Questions

Like the information on the Carnoy's fixative, this is a technique that allows you to see into the embryo better than with the live specimen. Do see Mitosis & Centrosomes!

After one treatment, the isolated mitotic apparatus looked very different than the above control. Note that the microtubules have grown longer than the cell could accommodate, resulting in a "spiral" array of microtubules.