H&E Staining and Stroke
At a glance
The death of brain cells, particularly neurons and oligodendrocytes, is central to stroke pathophysiology and treatment. Therefore, accurate observation of cell death in the brain is critical for diagnosing stroke, understanding mechanisms of tissue loss, and evaluating therapeutic strategies.
Fig.1. H&E staining of the cortex. (Kumar, et al., 2019)
Among numerous brain cell staining and labeling techniques, Ace Therapeutics performs Hematoxylin and Eosin (H&E) staining of stroke samples to help identify the different types of cells and tissues that are recognized and to provide important information about the pattern, shape, and structure of the cells in tissue samples.
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What is H&E Staining?
HE staining is a standard laboratory procedure that uses two dyes, hematoxylin and eosin, to show various parts of cells under the microscope. Hematoxylin is precise for determining nuclear structures, such as heterochromatin and nucleoli; eosin for cytoplasmic structures, such as collagen and elastic fibres, muscle fibres, and red blood cells. In high-quality H&E stains, the staining produces subtle differences in color tone, especially with eosin, which can help detect and interpret morphological changes associated with disease.
These two stains, together, will give you an idea of the general configuration of cells and their distribution and structure. This method has one main advantage: you can dye sections of tissue easily.
The Basic Steps of H&E Staining
Eliminate Wax
After preparing a paraffin section, the tissue is surrounded by hydrophobic paraffin wax, which prevents the visibility of cell and tissue components. To perform an H&E stain, the first step is to remove the wax using xylene, a hydrocarbon solvent.
Rehydrate the Section
After de-waxing the paraffin section, the slide is treated with multiple alcohol solutions to eliminate xylene, followed by a thorough rinse in water. This process hydrates the section, allowing aqueous reagents to penetrate the cells and tissue components effectively.
Stain the Nuclei with Hematoxylin
The slide is stained with a nuclear stain, typically Harris hematoxylin, which contains oxidized hematoxylin and an aluminum salt as a mordant. This staining process primarily colors the nuclei and some other components a reddish-purple hue.
Enhance Nuclear Staining with "Blueing"
After rinsing the section in tap water, it undergoes a "blueing" process with a weakly alkaline solution, which transforms the hematoxylin into a dark blue color. Following this, the section is rinsed again and evaluated to ensure the nuclei are properly stained with good contrast and to assess the level of background staining.
Differentiate to Remove Excess Background Stain
When using Harris hematoxylin, a differentiation step is often necessary to remove non-specific background staining and enhance contrast. This is achieved with a weak acid alcohol treatment. After differentiation, blueing and thorough rinsing are required again. Staining methods that involve this destaining process are called "regressive" stains.
Counterstain with Eosin
The section is stained with an aqueous or alcoholic solution of eosin, which imparts various shades of pink to many non-nuclear elements.
Rinse, Dehydrate, Clear, and Mount with Cover Glass
After staining with eosin, the slide is treated with several alcohol solutions to eliminate water, followed by rinsing in xylene to clear and make the tissue transparent. A thin layer of polystyrene mountant is then applied, followed by a glass coverslip. If performed correctly, the slide will display all important microscopic components and remain stable for many years.
- Kumar, G., et al. (2019). Neuroprotective effect of chlorogenic acid in global cerebral ischemia-reperfusion rat model. Naunyn-Schmiedeberg's Archives of Pharmacology, 392, 1293-1309.
