Collagen Preparation

1. Collect tails from adolescent rats, and rinse in 95% ethanol. Keep frozen or use fresh.

2. Sterilize frozen tails by rinsing in 95% ethanol, and remove the tendons as follows: Clamp the tail 2 cm from its distal end using a hemostat and clamp the second hemostat immediately adjacent and proximal. Fracture the tail by bending it sharply with the hemostats; the distal portion of the tail will now be held only by the tendons. Slide this piece of tail off the tendons slowly. Cut the pieces of tendon that dangle from the remainder of the tail, so that they drop into a sterile Petri dish. Repeat, working up the tail, until the tendons are completely removed.

3. Wash tendons three times with sterile water. Tease apart using blunt #5 forceps, so they form a fine mesh of fibers. Collect 3 g of wet tendons.

4. Dissolve for 24-36 h, but no longer (7), in 300 mL of 3% (v/v) glacial acetic acid at 4°C in a 200 mL sterile conical flask, stirring as slowly as possible (7) (note that the stir bar will stop stirring as the solution becomes viscous; check a few times during first few hours).

5. When most of the tendons have dissolved, spin at 20,000g for 60 min to pellet the remainder.

6. Transfer supernatant to dialysis tubing (boiled in EDTA and washed extensively in sterile water), and dialyze at 4°C against 3 L of sterile 0.1X DMEM, pH 4.0, without bicarbonate. Dialyze for three days, changing medium once a day. Dialysis removes excess acid while keeping the pH low (the collagen gels when alkalinized).

7. Store in 15-50-mL aliquots at 4°C. Do not freeze. Keeps at least 6-12 mo.

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