Haematoxylin and Eosin 101: Part Two- Recipes and Materials

Following on from the first part of the H and E 101 articles, here are the materials and recipes you’ll need for your own H and E workstation (assuming you don’t have access to a histology lab).

Many of the chemicals listed below are toxic and/or harmful. Use PPE when handling/storing, follow SOP’s in your own institute and use the COSHH and MSDS forms.

Here are the four solutions you’ll need (plus an easy guide to the gradated alcohols);

(1)  Harris’ Haematoxylin Solution.

Haematoxylin                                                             2.5 g

Ethanol                                                                      25 ml

Potassium alum (the ‘mordant’)                                 50 g

Deionised water                                                       500 ml

Sodium iodate                                                          0.5 g

Acetic acid (glacial)                                                  20 ml

The haematoxylin needs to be oxidized to become a functional dye- this is achieved by the addition of sodium iodate. The oxidized haematoxylin (‘haematein’) then binds to the mordant which allows it to bind to nuclear material.

Haematoxylin is dissolved in ethanol, and is then added to the potassium alum, which has previously been dissolved in 500 ml of warm deionised water in a two litre flask.

The mixture is rapidly brought to the boil and the sodium iodate is then slowly and carefully added. Cool the solution by plunging the flask into cold water or into a sink containing chipped ice. When the solution is cold, add the acetic acid and the stain is ready for immediate use. The glacial acetic acid is optional, but its inclusion gives more precise and selective staining of nuclei.

Filter before use and replace every two weeks.

(2)  Acid Alcohol.

Hydrochloric acid, concentrated (sp.gr.1.19)      1 ml

70% ethanol                                                         99 ml

(3)  Scotts Tap Water.

Sodium bicarbonate                                                           2 g

Magnesium sulphate                                                          20 g

Deionised water                                                                1 litre

Dissolve the salts in the water.  Store stock solutions at room temperature.

(4)  Eosin.

Eosin Y is made up as a 1 % (w/v) solution in dH₂0 (ie- 1 g of Eosin Y in 100 ml of water).

Alcohol gradient

95% ethanol

Ethanol (GP grade)                                     95 ml

Deionised water                                           5 ml

80% ethanol

Ethanol (GP grade)                                     80 ml

Deionised water                                           20 ml

70% ethanol

Ethanol (GP grade)                                     70 ml

Deionised water                                           30 ml

Eosin Y solution can be bought commercially and it’s an easier option than weighing dye powder. Haematoxylin solution can also be bought as a ready-made solution, but will depend if you plan to use gallons of it!

Happy H and E staining!