Neuromelanin quantification in formalin-fixed substantia nigra and locus coeruleus
Dipshay Avi Chand, Miriam Scadeng, Victor Dieriks
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Abstract
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.
The last step contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.
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
Neuromelanin synthesis
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
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).
Stop the reaction by decreasing the solution pH to between 3 and 4, using 33% acetic acid.
Keep the solution wrapped in foil, as before. Incubate for 16 hours at 95°C, in an oven.
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.
Add 1.5 mL Tris buffer and incubate for 2 hours at 37°C.
Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.
Add 1.5 mL sodium chloride solution and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.
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.
Add 1.5 mL acetone and wash the pellet. Centrifuge the tube for 2 minutes at 9000 x g. Pour off supernatant.
Dry the neuromelanin under flowing nitrogen gas for about 24 hours.
Store the synthetic neuromelanin at -20°C in a closed plastic tube that is wrapped with aluminium foil to prevent light exposure.
Create a neuromelanin stock solution
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.
Quantify neuromelanin in a brain region
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.
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.
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.
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.
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).
Centrifuge the tube for 10 minutes at 12,000 x g.
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.
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.
Remove 450 μL of the supernatant. Add 950 μL ultrapure water.
Centrifuge the tube for 10 minutes at 12,000 x g.
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.
Incubate for 1 hour at 80°C. Shake halfway through the incubation period.
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.
Neuromelanin calibration curve
Incubate in a dry heating block for 1 hour at 80°C. Centrifuge for 5 minutes at 15,000 x g.
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.
Measure the absorbance at 350 nm using an absorbance spectrophotometer. Use 2 M sodium hydroxide as the blank solution.
Spotlight video
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