4.6. Sample preparation

1. Remove the stored brains from the freezer or liquid nitrogen and place them in a covered ice bucket.

2. Add 20 µl of chilled IS-B solution to each tube.

3. Homogenize each brain using a separate tissue grinder.

4. Remove the tissue and liquid from the pestle to ensure that all tissue remains in the solution.

5. Suspend the sample tubes in an ultrasonic bath filled with ice water.

6. Cover to reduce light exposure.

7. Sonicate the tubes for 5 min to disrupt tissue.

8. Leave the tubes covered and in ice water for an additional 20 min to allow the amines to be extracted from the brain tissue.

9. Centrifuge the tubes at 12,000 RCF for 10 min at 4 °C.

10. Collect the supernatant and pass it through a 0.22-μm nylon membrane filter into a fresh, labelled microcentrifuge tube.

This step will help remove any remaining tissue, protecting the HPLC system from clogging.

11. Store the tube containing the supernatant on ice and protected it from light until used.

12. Place the original tube containing the residual brain tissue in a -80 °C freezer for later quantification.