Melanin Stain (Ferrous Sulfate Method)

Melanin Stain (Ferrous Sulfate Method)

Melanin Stain


The adsorption of ferrous ions using melanin Stain and the subsequent formation of melanin ferrous complex play a crucial role in various biological processes. This document outlines a detailed method for this ferrous ion adsorption technique, which also involves Turnbull’s Blue reaction. The procedure employs several reagents and solutions to achieve accurate results.

Main Components and Reagent Composition

The following are the main components of the method:

  • Ferrous Sulfate Solution: This solution contains ferrous sulfate and is used during the initial phase of the process.
  • Potassium Ferricyanide Acetic Acid Solution: A critical component of the reaction is potassium ferricyanide and glacial acetic acid.
  • Glacial Acetic Acid Solution: Used for washing purposes, this solution contains glacial acetic acid.
  • Van Gieson Solution: The Van Gieson stain consists of acid fuchsin and picric acid.

Sample Requirements and Test Procedure

Prior to the test, the tissue section must be adequately fixed. The test procedure is as follows:

  • Dewax the slices routinely with water and then wash them once with distilled water.
  • Introduce the Ferrous Sulfate Solution and allow it to react for 1 hour.
  • Perform 3 washes with distilled water, each lasting approximately 3 to 5 minutes.
  • Add the Potassium Ferricyanide Acetic Acid Solution and let it react for 30 minutes.
  • Wash the samples slightly with the Glacial Acetic Acid Solution for 2 to 3 seconds and then allow them to dry.
  • Stain the samples with the Van Gieson Solution for about 1 minute.
  • After applying the Van Gieson stain, promptly differentiate with 95% alcohol for several seconds.
  • Dehydrate and make the samples transparent using conventional methods. Finally, seal them with neutral resin.

Result Interpretation

The outcome of the procedure can be interpreted based on the following color codes:

  • Melanin: Green or dark green
  • Collagen fibers: Red
  • Muscle fibers: Yellow

Recommendations and precautions

Precautions should be taken to ensure accurate results and safe handling of reagents:

  • It is advisable to fix the samples with formaldehyde or Carney solution, as chromate fixatives should be avoided.
  • In cases where hemosiderin shows a green reaction, additional iron staining is necessary to confirm the presence of ferrous ions.
  • Rapidly differentiate the samples with 95% alcohol after Van Gieson staining to prevent discoloration.
  • Avoid washing the Van Gieson Solution after staining, as it may weaken or elute the color.
  • This method should be executed by professionals who have carefully read the instruction manual and practiced proper personal hygiene protection.
  • Dispose of waste in accordance with the hospital or environmental protection department regulations.
  • The production date, production batch number, and expiration date should be visible on the outer packaging.


  • Chinese Medical Association. Clinical Technical Operating Specifications – Pathology Volumes. People’s Military Medical Publishing House.
  • Ling Qibo. Practical Pathology Special Staining and Histochemistry Technology. Guangdong Higher Education Press.


The melanin ferrous complex formation and Turnbull’s Blue reaction offer a valuable technique for ferrous ion adsorption. By following the specified procedure and adhering to the precautions, researchers and professionals can achieve accurate and reliable results in various biological applications.

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