The vitelline envelops are removed with Dumont #5 watchmakers forceps. For cutting embryos, we use eyebrow hairs embedded in wax in the tips of pulled, disposable "Pasteur" pipets. Hairloops, which are used to position and hold embryonic tissues, are also embedded in pipets. Pipets are pulled to a diameter several times that of a hair and scored with a diamond pencil; the vibration of scoring will usually fracture the pipet transversely. An eyebrow hair with a fine, evenly tapered point is placed in the pipet, butt first, such that its natural curvature brings the distal third to an angle of about 120° with respect to the pipet. In order to make a hairloop, both ends of a long hair are placed into the pipet until the smallest possible loop is formed. (It is important to use a long piece of hair, since the friction of the hair on the side of the pipet, a key factor in making a small loop, is increased with length.) Pipets are then dipped in wax, which has been melted in a spoon. The excess wax is removed by heating a steel spatula in an alcohol burner until warm, covering it with small piece of laboratory tissue, and transiently touching the eyebrow hair or hairloop to the tissue. Once melted, the excess wax will soak into the tissue without releasing the eyebrow hair or hairloop from the pipet. These instruments are sterilized by dipping them in 70% ethanol for a few minutes. We have found that these "low-tech" instruments outperform more pretentious instruments; they are sharp, flexible, and relatively nonadhesive to cells.
For microsurgery, a plastic disposable 60-mm Petri dish is used. Grafts between embryos are most easily made in a dish containing black modeling clay (Pastalina, Certified Nontoxic, Van Aken International, Rancho Cucamonga, CA 91730). The clay, available from many toy and art stores, is pressed into the bottom of the dish to a depth of 2-3 mm. To sterilize the clay, the dish is flooded for 20-30 s with 70% alcohol and rinsed in five changes of sterile saline.
To hold grafts or explants in place, small fragments of coverslips are cut ahead of time in rectangular shapes, about 2-4 mm by 10-15 mm, with a diamond pencil, and kept in plastic Petri dishes. These coverslip fragments are handled with watchmaker's forceps (see Subheading 3.6.).
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