TITLE: Neuromelanin quantification in formalin-fixed substantia nigra and locus coeruleus
AUTHORS: Dipshay Avi Chand, Miriam Scadeng, Birger Victor Dieriks

[DESCRIPTION]
There are limited methods for absolute quantification of neuromelanin in brain regions such as the substantia nigra and locus coeruleus. While stereology is typically used to quantify neuromelanin in these regions, it is semi-quantitative.

A protocol for absolute quantification of neuromelanin using spectrophotometry was adapted for the modern laboratory. The process was improved so neuromelanin could be reliably quantified in formalin-fixed samples. A methodology to synthesise neuromelanin is also described, which prevents the wastage of substantia nigra neuromelanin since it is required for the calibration curve. The methodology described here is unbiased, high-throughput, and can measure neuromelanin concentration reliably.

[BEFORE_START]
This protocol will use an absorbance spectrophotometer that can measure volumes less than 200 μL. 

Prepare all required buffers and solutions before starting (see materials section).

If you already have dried neuromelanin (synthetic or endogenous), skip to step 13.

[STEPS]
SECTION: Neuromelanin synthesis
1. 
 


Add 50 mL phosphate buffer, 630 μL  dopamine stock solution, 61 μL L-cysteine stock solution,  and 250 μL mushroom tyrosinase stock to an Erlenmeyer flask

SECTION: Neuromelanin synthesis
2. Wrap the flask with aluminium foil and make small holes on top; this protects the reaction from light but allows exposure to air. Put the flask in an incubator for 48 hours, at 37°C, with constant shaking (120 rpm).

 

SECTION: Neuromelanin synthesis
3. Stop the reaction by decreasing the solution pH to between 3 and 4, using 33% acetic acid.
SECTION: Neuromelanin synthesis
4. Keep the solution wrapped in foil, as before. Incubate for 16 hours at 95°C, in an oven.

 

SECTION: Neuromelanin synthesis
5. Transfer the solution into 50 mL polypropylene tubes. Centrifuge the solution for 15 minutes at 3800 x g. Pour off the supernatant and transfer the slurry at the bottom into a 2 mL microcentrifuge tube.



 


SECTION: Neuromelanin synthesis
6. Add 1.5 mL Tris buffer and incubate for 2 hours at 37°C.

SECTION: Neuromelanin synthesis
7. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.
SECTION: Neuromelanin synthesis
8. Add 1.5 mL sodium chloride solution and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.

SECTION: Neuromelanin synthesis
9. Add 1.5 mL ultrapure water and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.

Repeat this step once more.
SECTION: Neuromelanin synthesis
10. Add 1.5 mL acetone and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.

 

SECTION: Neuromelanin synthesis
11. Dry the neuromelanin under flowing nitrogen gas for about 24 hours.

 

SECTION: Neuromelanin synthesis
12. Store the synthetic neuromelanin at -20°C in a closed plastic tube that is wrapped with aluminium foil to prevent light exposure.

SECTION: Create a neuromelanin stock solution
13. 
 

Add the frozen, dried neuromelanin to ultrapure water, adjust to pH 9.0. Sonicate in an ultrasonic ice water bath for 20 minutes. Store this solution at 4°C until use.
SECTION: Quantify neuromelanin in a brain region
14. 
 

Weigh 2-20 mg of fixed brain tissue (substantia nigra or locus coeruleus) using a high-accuracy scale. Homogenise the tissue in a 1.5 mL tube, using a mini plastic pestle, until a relatively fine homogenate is formed. 

 

SECTION: Quantify neuromelanin in a brain region
15. Add 1000 μL tissue lysis buffer, cap tubes, and incubate in a dry heating block for 90 minutes at 90°C. Shake tube halfway through incubation.

Centrifuge the tube for 10 minutes at 15,000 x g.


SECTION: Quantify neuromelanin in a brain region
16. Remove 900 μL of the supernatant using a micropipette, leaving behind 100 μL in the tube. Add 900 μL phosphate buffer.

Centrifuge the tube for 10 minutes at 15,000 x g.
SECTION: Quantify neuromelanin in a brain region
16.1. Create the Proteinase K digestion solution by adding Proteinase K to Tris buffer to achieve a concentration of 0.25 mg/mL Proteinase K. Keep solution on ice.

SECTION: Quantify neuromelanin in a brain region
17. Remove 900 μL of the supernatant. Add 900 μL Proteinase K digestion solution. Incubate the tube overnight (12-14 hours) at 50°C, with constant shaking (130 rpm).

 

SECTION: Quantify neuromelanin in a brain region
17.1. Centrifuge the tube for 10 minutes at 12,000 x g.

SECTION: Quantify neuromelanin in a brain region
18. Remove 900 μL of the supernatant, leaving behind 100 μL in the tube. Add 900 μL sodium chloride solution.

Centrifuge the tube for 10 minutes at 12,000 x g.

 

SECTION: Quantify neuromelanin in a brain region
19. Remove 950 μL of the supernatant, leaving behind 50 μL. Add 450 μL acetone.

Wait for 5 minutes.
Vortex the tube for ~5 seconds.
Place in a room-temperature ultrasonic water bath for 3 minutes.

Centrifuge the tube for 10 minutes at 12,000 x g.
SECTION: Quantify neuromelanin in a brain region
20. Remove 450 μL of the supernatant. Add 950 μL ultrapure water.

Centrifuge the tube for 10 minutes at 12,000 x g.
SECTION: Quantify neuromelanin in a brain region
21. Remove 900 μL of the supernatant, leaving behind 100 μL. Add 100 μL 4 M sodium hydroxide.
The final volume should be 200 μL. 

Sonicate the tube in a room-temperature ultrasonic water bath for 5 minutes.

SECTION: Quantify neuromelanin in a brain region
22. Incubate for 1 hour at 80°C. Shake halfway through the incubation period.

 

SECTION: Quantify neuromelanin in a brain region
23. Centrifuge the tube for 10 minutes at 15,000 x g.

Measure the absorbance at 350 nm using an absorbance spectrophotometer; we used the NanoDrop One (Thermo Fisher Scientific). Use 2 M sodium hydroxide as the blank solution. 


SECTION: Neuromelanin calibration curve
24. 
 

In triplicate, add 160 μL of the 0.5 mg/mL neuromelanin stock solution, 40 μL ultrapure water, and 200 μL 4 M sodium hydroxide into a  1.5 mL tube. This is a 200 μg/mL solution.

Incubate in a dry heating block for 1 hour at 80°C. Centrifuge for 5 minutes at 15,000 x g.

SECTION: Neuromelanin calibration curve
25. Starting with the 200 μg/mL solution, use two-fold serial dilutions to create solutions that are 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL. Use 2 M sodium hydroxide as the diluent.
SECTION: Neuromelanin calibration curve
26. Measure the absorbance at 350 nm using an absorbance spectrophotometer. Use 2 M sodium hydroxide as the blank solution.