Cutting Tissues

Large pieces that are to be cut along straight lines from an intact embryo are cut out by pressing an eyebrow hair directly through the embryo, along the line of the cut until the substratum is reached. Explants are trimmed to size with the same type of movement (Fig. 4A). It is best to use a long, stiff eyebrow hair for this type of operation. In order to remove only a superficial part of the embryo without damaging deeper tissues, a type of "stitching motion" is used. The eyebrow hair is inserted into the wall of the embryo at an angle of 45° with the surface until the tip is at the desired depth; the hairloop is placed next to the eyebrow hair, along the line of cut on the surface of the embryo, and the tissue is cut by quickly raising the tip of the eyebrow hair (Fig. 4B). The operation is repeated very quickly, advancing the eyebrow hair and the retaining hairloop only a small distance at each cycle, never cutting through more than a few cells at a time, to avoid undue strain in the tissue (Fig. 4B). By varying the depth of insertion of the eyebrow hair, a cut of any depth can be made, ranging from the epithelial layer alone (about 10-15 ^ thick) to the epi-

Fig. 4. Methods of cutting (A,B), peeling (C), and shearing cells off layers (D) are shown with diagrams. In order to trim an explant usin a long, straight type of cut (A), the tissue is held lightly with a hairloop, while a long, stiff eyebrow hair knife is brought down on the explant along the desired line of the cut (dashed line, A, left), and pressed through the tissue until it hits the substratum. The eyebrow hair is then quickly moved to the right, cleanly separating the two pieces of tissue (A, right). A method of cutting through a limited and controlled depth of tissue is shown in a face-on view of the cutting surface, as if the tissue on the viewer's side of the cut had been removed (B). The hairloop is used to hold the tissue along the line of the cut, and the eyebrow hair tip is inserted to the desired depth and lifted repeatedly and quickly (arrows, B), cutting only a few cells at a time. A method of peeling tissue from underlying tissue is shown (C). In this diagram, the postinvolution PM (below restraining hairloop) and the neural ectoderm (above the eyebrow hair) of an early gastrula are being separated. The tip of the eyebrow hair is moved along the interface between the tissues (solid arrow), whereas the heel of the curved hair is rocked against the prospective neural tissue (open arrows). Adherent mesodermal cells are sheared off from the inner surface of an explant of the neural tissue (D). The hairloop is placed at the bottom of the tissue to prevent the tissue from moving toward the operator. The eyebrow hair is then swept at low angles straight toward the operator, just above the surface of the explant, shearing off the adherent cells. For clarity, the size of the eyebrow hair and hairloop is reduced relative to the size of the tissues.

thelial and deep region together (about 25-100 pm, depending on the stage and region). A small, sharp-tipped eyebrow hair is best for this type of stitching cut. An alternative method for cutting tissue at a limited depth is to insert a long, stiff eyebrow hair along a line of tissue that is to be cut, and then rub the hairloop back and forth against it, on the outside, cutting the intervening tissue. This method is good for making straight cuts, but is not conducive to making curved cuts, and it does not allow precise control of depth below the curved surface of the embryo.

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