Mar 27, 2023

Neuromelanin staining (Fontana-Masson staining)-DAB staining on midbrain organoids

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx98mdg8j/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx98mdg8j/v1
Protocol Citation: michela.deleidi , Maria Jose Perez J., Hariam Raji, Pascale Baden, Federico Bertoli 2023. Neuromelanin staining (Fontana-Masson staining)-DAB staining on midbrain organoids. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx98mdg8j/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 17, 2023
Last Modified: March 27, 2023
Protocol  Integer ID: 78956
Keywords: Fontana-Masson Stain Kit, Neuromelanin staining , staining technique, melanin, th staining, dab, antibody, antigen, containing cell, cell, colored product
Abstract
Fontana-Masson staining is a silver staining technique that is commonly used to identify melanin-containing cells. That was combined with DAB-TH staining that works by using an antibody to detect the presence of TH, followed by a reaction with a substrate (DAB) that results in the formation of a brown-colored product at the site of the antigen-antibody interaction.
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Materials
Materials

  • methanol (MeOH)
  • 3% hydrogen peroxide
  • PBS
  • Triton-X 0.2%
  • DAB solution
  • VECTASTAIN® Elite® ABC-HRP Kit
Fontana-Masson Stain KitMerck MilliporeSigma (Sigma-Aldrich)Catalog #HT200-1KT

Neuromelanin staining
5h 35m 30s
Incubate human midbrain organoid sections in a fresh solution of 3:1 methanol (MeOH)/3% hydrogen peroxide at Room temperature for 00:20:00 .

20m
Wash the slides and block it.
Wash the slides in PBS for 00:05:00 . (1/3)

5m
Wash the slides in PBS for 00:05:00 . (2/3)
5m
Wash the slides in PBS for 00:05:00 . (3/3)
5m
Then, block with NGS 10% in PBS+ Triton-X 0.2% for 01:00:00 at Room temperature .

1h
Apply primary antibodies in NGS 5% in PBS+ Triton-X 0.2% solution Overnight at 4 °C .

1h
Next, wash slides.
Wash the slides in PBS for 00:05:00 . (1/3)
5m
Wash the slides in PBS for 00:05:00 . (2/3)
5m
Wash the slides in PBS for 00:05:00 . (3/3)
5m
Apply secondary antibodies to NGS 5% in PBS+ Triton-X 0.2% solution for 01:00:00 at Room temperature .
1h
Prepare ABC solution from Vectastain according to the manufacturer’s instructions (VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard) PK-6100) and apply to sections for 01:00:00 at Room temperature .
1h
Wash the slides.
Wash the slides in PBS for 00:05:00 . (1/3)
5m
Wash the slides in PBS for 00:05:00 . (2/3)
5m
Wash the slides in PBS for 00:05:00 . (3/3)
5m
Prepare DAB solution according to the manufacturer’s instructions by diluting in 50 mL of 1x PBS with 50 µL of 3% H2O2.

Apply DAB solution to the sections at Room temperature for 30 seconds to 00:12:00 depending on when the visible reaction occurred.

12m
For the visualization of neuromelanin, use the Fontana-Masson stain kit, according to the manufacturer’s instructions. (Fontana-Masson Stain Kit; Sigma‒Aldrich-HT200).
Eventually mount slides with synthetic resin.