Mar 27, 2023

Public workspaceNeuromelanin staining (Fontana-Masson staining)-DAB staining on midbrain organoids

DOI
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: dx.doi.org/10.17504/protocols.io.kxygx98mdg8j/v1
Protocol Citation: michela.deleidi, María José Pérez 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
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
ReagentFontana-Masson Stain KitMerck MilliporeSigma (Sigma-Aldrich)Catalog #HT200-1KT

Neuromelanin staining
Neuromelanin staining
5h 35m 30s
5h 35m 30s
Incubate human midbrain organoid sections in a fresh solution of 3:1 methanol (MeOH)/3% hydrogen peroxide at TemperatureRoom temperature for Duration00:20:00 .

20m
Incubation
Wash the slides and block it.
Wash
Wash the slides in PBS for Duration00:05:00 . (1/3)

5m
Wash the slides in PBS for Duration00:05:00 . (2/3)
5m
Wash the slides in PBS for Duration00:05:00 . (3/3)
5m
Then, block with NGS 10% in PBS+ Triton-X 0.2% for Duration01:00:00 at TemperatureRoom temperature .

1h
Apply primary antibodies in NGS 5% in PBS+ Triton-X 0.2% solution DurationOvernight at Temperature4 °C .

1h
Overnight
Next, wash slides.
Wash
Wash the slides in PBS for Duration00:05:00 . (1/3)
5m
Wash the slides in PBS for Duration00:05:00 . (2/3)
5m
Wash the slides in PBS for Duration00:05:00 . (3/3)
5m
Apply secondary antibodies to NGS 5% in PBS+ Triton-X 0.2% solution for Duration01:00:00 at TemperatureRoom 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 Duration01:00:00 at TemperatureRoom temperature .
1h
Wash the slides.
Wash
Wash the slides in PBS for Duration00:05:00 . (1/3)
5m
Wash the slides in PBS for Duration00:05:00 . (2/3)
5m
Wash the slides in PBS for Duration00:05:00 . (3/3)
5m
Prepare DAB solution according to the manufacturer’s instructions by diluting in Amount50 mL of 1x PBS with Amount50 µL of 3% H2O2.

Apply DAB solution to the sections at TemperatureRoom temperature for 30 seconds to Duration00:12:00 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.