In hematoxylin-eosin staining (H&E), the most widely used histological staining technique, nuclear staining is achieved using ready-to-use staining solutions, such as Mayer’s hemalum solution or Hematoxylin solution modified according to Gill III. 

These solutions do not need to be filtrated prior use and the dye is oxidized in a controlled manner, which renders the results robust and allows a brilliant differentiation of the stained structures.

Counterstaining of i.e. for proteins, collagen, keratin, intercellular substances. can be performed with alcoholic or aqueous Eosin Y solution 0.5 %. It is registered as IVD and CE certified product and can be used for clinical diagnostics.

Mayer’s hemalum solution Overview

• Store the Mayer’s hemalum solution – for microscopy at +15 °C to +25 °C.
• Dye precipitation in the staining solutions might occur with storing temperatures below +15 °C. In that case the bottles should be put into a water bath of approx. 60 °C for 2 – 3 hours. The solutions should be filtered before use.
• The Mayer’s hemalum solution – for microscopy can be used until the stated expiry date. After first opening of the bottle, the contents can be used up to the stated expiry date when stored at +15 °C to +25 °C. The bottles must be kept tightly closed at all times.
• In order to avoid errors, the application must be carried out by qualified personnel only.

Mayer’s hemalum solution Application

• This “Mayer’s hemalum solution – for microscopy” is used for human-medical cell diagnosis and serves the histological and clinico-cytological investigation of sample material of human origin. 
• It is a staining solution that when used together with other in vitro diagnostic products from our portfolio makes target structures evaluable for diagnostic purposes (by fixing, embedding, staining, counterstaining, mounting) in histological and clinico-cytological specimen materials, for example histological sections of e. g. the kidney, muscle tissue, heart, or lung.
• The hematoxylin and eosin (H&E) staining method is the method most frequently used for the staining of histology material. This Mayer’s hemalum solution is used in the hematoxylin-eosin (H&E) overview staining procedure, a method routinely used in histology.

Principle

The hematoxylin and eosin (H&E) staining method is the method most frequently used for the staining of histology material. The staining mechanism is a physico-chemical process. In the first step, the positively charged nuclear dye (hematoxylin) binds to the negatively charged phosphate groups of the nucleic acid of the cell nucleus. The nuclei will be dyed dark blue to dark violet.

The second step is the counterstaining with negatively charged anionic xanthene dye (eosin Y, eosin B, or erythrosine B). This binds to the positively charged plasma proteins. Cytoplasma and intercellular substances are stained pink to red, while erythrocytes will appear with yellow to orange color.

Two methods can be distinguished. With the progressive method staining is carried out to the desired intensity, followed by the bluing step in tap water to make colour permanent. With the regressive method the material is over-stained and the excess of staining solution is removed by acid rinsing steps, followed by the bluing step to make color permanent. The structures of nuclei are more differentiated and better visible by the regressive method.

Components Require

The following components are required to prepare 1L of Mayer’s Hemalum Solution.

Component	Amount	Concentration
aluminum potassium sulfate (KAl[SO4]2•12H2O) (mw: 474.39 g/mol)	50 g	0.1053 M
hematoxylin (C.I. 75290) (mw: 302.28 g/mol)	1 g	0.0033 M
sodium iodate (NaIO3) (mw: 197.89 g/mol)	100 mg	0.0005 M
citric acid (monohydrate) (mw: 210.14 g/mol)	1 g	0.0048 M
chloral hydrate (mw: 165.4 g/mol)	50 g	0.3022 M

Mayer’s hemalum solution Preparation 

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 50 g of aluminum potassium sulfate (KAl[SO4]2•12H2O) to the solution.
3. Add 1 g of hematoxylin (C.I. 75290) to the solution.
4. Add 100 mg of sodium iodate (NaIO3) to the solution.
5. Add 1 g of citric acid (monohydrate) to the solution.
6. Add 50 g of chloral hydrate to the solution.
7. A magnetic stirrer will hasten dissolving of the alum, hematoxylin, and hematein formed by iodate oxidation.
8. Add distilled water until volume is 1 L.
9. Mayer’s hemalum often keeps for 1 yr but some batches lose their potency after only a few months. The solution may be reused many times.

References

1. “Mayer’s Hemalum Preparation.” AAT Bioquest, Inc, 02 Jun. 2021, https://www.aatbio.com/resources/buffer-preparations-and-recipes/mayer-s-hemalum.
2. https://www.sigmaaldrich.com/catalog/product/mm/109249?lang=en&region=IN