Dissection of Donor Notochords

To explant the notochord from donor embryos, use one of the three methods described below. Method 1 is a rapid procedure, but may leave cells from adjacent tissues sticking to the explant. Methods 2 and 3 use proteolytic enzymes to free the notochord from adhering cells, thus providing clean grafts. The best results are obtained with method 3. Here, the embryos are treated very gently to maintain their structural integrity and to recover additional tissues for further transplantation experiments.

3.3.1. Mechanical Method

1. Cut off and discard body from HH 13-16 embryos below the level of the last somites.

2. Grip the embryo with two pairs of forceps, one squeezing around the first branchial arch, and the other gripping the upper trunk. Pull apart. The notochord will stay attached to the head and will slide out intact from the trunk.

3. Trim off the notochord, and set it aside in a dish of fresh Howard's Ringer on ice (see Note 3).

3.3.2. Enzymatic Method 1

1. Transfer embryo to a Sylgard-bottomed dish containing Howard's Ringer. Submerge and pin out through the membranes with stainless-steel minuten pins. Subdissect region using needles or ultrafine scissors under transmitted illumination.

2. Using a serum-coated 20-|jL tip, transfer tissues to a solution of 1 mg/mL Dispase, 5 ^g/mL DNase I in L15 medium in a 35-mm dish.

3. Treat for 20-25 min at room temperature, and then transfer to fresh Howard's Ringer containing 10% serum. Reflux notochord through tip until free of adherent cells—check under stereoscope. Use fine needles to help separation if necessary.

4. Transfer to fresh Howard's Ringer, and set on ice (see Note 3).

3.3.3. Enzymatic Method 2

1. Pin down the embryo as in Subheading 3.2.2. The notochord is reached best if the embryo is ventral side up.

2. Carefully open the embryo along the notochord, using the sharpened tungsten needles.

3. Overlay the embryo with 50-100 ||L Dispase solution. Leave at room temperature until structures come loose (15-20 min). Use operating needles to help separation.

4. Cut notochord with needles or ultrafine scissors.

5. Transfer to fresh Howard's Ringer on ice (see Note 3).

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