Notes

1. These gonadotropins lose their activity upon thawing and should be kept on ice until just prior to use.

2. This strain responds well to superovulation with 30-40 embryos per animal under the most optimal conditions. Mice should be ordered at 3 wk of age but allowed to adjust to their surroundings for 4-5 d prior to superovulation..

3. Care should be taken to keep the pH of the fixative between 6.0 and 7.0.

4. It is important to transfer the embryos in the smallest possible volume to avoid diluting prior conditions. It is also necessary to follow the time periods suggested for fixation and permeabilization, as longer times will allow the embryos to firmly adhere to the plastic and forceful removal may cause destruction of the embryos.

5. Care should be taken in mounting the embryos. A small droplet, 4-5^L, of Vec-tashield is added to the cover slip, and the coverslip is inverted with the droplet directly over the PAP well. Too much Vectashield will cause the embryo to float out and over the well if it is not firmly adherent. Too little mounting solution will cause bleaching to occur and may cause visualization to be impossible. Often it is helpful to redraw the encircling PAP pen well just prior to the placement of the cover slip to avoid losing the embryos.

6. This technique allows the mouse to serve as its own control and is somewhat easier than the whole-animal fixation technique. The problem with this alternative method, however, is that the muscle dissection occurs before fixation, and if the muscle contracts during this dissection, translocation may occur regardless of insulin stimulation. This contraction-induced translocation may inadvertently drive the GLUT-4 to the plasma membrane and thus interfere with detection of a difference in location between the transporter in a basal versus insulin-stimulated state.

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