To begin, apply a rectangular outline of clear nail polish to the glass slide. Prepare several such transplant slides. For mounting the anesthetized animal, use a Pasteur pipette and pump to transfer it from RPT to low-melting agarose or LMA.
Then, transfer the animal in a small drop of LMA onto the slide within the nail polish outline. Before the LMA solidifies, use an embryo pusher to orient the animal vertically, with the intended needle insertion site to the right. And let the agarose fully set for approximately five minutes.
using a scalpel, cut a straight vertical slice through the agarose drop just to the right of the mounted animal. Wipe off loose agarose from the slide. Employing a scalpel, cut wedges out of the agarose to expose the needle insertion site.
Apply RPT to the slide until the agarose drops are fully submerged. And mount the prepared slide on the transplantation microscope. Lower the 40X objective until it breaks the surface of the media and raise it to bring the donor animal into focus.
Then, bring the fluorescently labeled donor cells of interest into focus. Move the stage to the left in preparation to locate the transplant needle.
