Practical 2: Haematoxylin and Eosin Stain

Question 1

How wide are your microtomy slices

Answer 1

4 to 5 microns

Question 2

Describe the H&E stain

Answer 2

Aluminium-mordanted haematoxylin stain the nuclei

Proteins are stained varying shades of pink/red with the acidic dye Eosin

Question 3

How do you use your haematoxylin stani

Answer 3

The basic dye is applied and stains regressively -> negatively charged groups in tissue will stain but the nucleus will be more intense

The section will have a pink/purple colour before blueing

Question 4

Why do we need a differentiation step?

Answer 4

Allows for the selective removal of excess dye from components which have weekly bound the dye, resulting in the nuclei remaining stained and the cytoplasm clear

Must be checked microscopically to avoid over or under-differentiation

Question 5

How does blueing work

Answer 5

The pH of haematoxylin is low and the dye is pink

Washing in tap water raises the pH and changes the dye absorption so that it appears blue under the microscope

Question 6

How do you stain with Eosin
(3)

Answer 6

Eosin is an acid dye which will stain all components unstained by haematoxylin

It is a progressive stain as it does not require differentiation

It is a counterstain to haematoxylin

Question 7

Why do you need to dehydrate your slide

Answer 7

So that the slide can be placed in an organic solvent (xylene) and cover-slipped in a resin that solidifies and is a high refractive index and so it can be stored indefinitely

Question 8

What should you do to imprints and frozen tissue before staining

Answer 8

Fix sections in spirit