Why eosin stains cytoplasm

Most proteins in the cytoplasm are basic, and so eosin binds to these proteins and stains them pink. This includes cytoplasmic filaments in muscle cells, intracellular membranes, and extracellular fibres. Tissue stained with haematoxylin and eosin shows cytoplasm stained pink-orange and nuclei stained darkly, either blue or purple. Eosin also stains red blood cells intensely red. Progressive staining — When tissue is left in the stain just long enough to reach the proper end point.

The slides have to be examined at different interval to find out when the staining is optimum. So, when optimizing the stain, make sure to only edit the time of one of the components. This technique will help eliminate the need to spend additional time adjusting the stain.

With regressive and modified progressive staining, a differentiator is used. If the differentiator is made in-house, there is the potential for it to be either too weak or too strong. Both scenarios will impact staining. If the differentiator is stronger than intended, it will remove more hematoxylin and will make the nuclei pale.

Time is also important. Too much time in a properly prepared differentiator will also remove more hematoxylin and will ultimately understain the nuclei. Mild acidity is critical to the shelf life of hematoxylin. Without it, the alkalinity of the tap water rinse will raise the pH such that the dye lake can precipitate, and the color will change from cherry red to purple red. Adding small amounts of acetic acid to the hematoxylin periodically will aid in maintaining appropriate pH and can extend the life of the stain.

Water is used as a differentiator for eosin. The ethanol aids with rinsing the slide, while water pulls excess eosin from the tissue.

This step can help with coloration control but extending the time provides for lighter stains, while shortening the time maintains brighter coloration.

However, excess water in xylene can continue the differentiation process and can be seen after coverslipping as a pink haze on the slide. Not all tissues are created equally. Cysts and fatty samples, even when processed correctly, may be very difficult to see grossly once the slide has been stained.

These samples often have open spaces where fluids or fat were in the cell, and the thinness of the cell walls may give the appearance of being light when the coloration is simply an artifact of the tissue type. Highly cellular samples e. Remember that lymphocytes have little cytoplasm, and there is not nearly the cellular material between cells as with other tissues. For this reason, the hematoxylin does not have to compete with the eosin.

The compact nature of the cells also concentrates the DNA, giving these highly cellular tissues the appearance of being overstained, when in reality, they may simply need to be sectioned thinner.

The use of clean and fresh dewaxing reagents is essential for the removal of paraffin from the slide prior to the addition of the dyes. While xylene is the most commonly used solvent, xylene substitutes are gaining in popularity because they are considered less hazardous and more ecofriendly.

Water in solvents, whether from reagent contamination or a high humidity environment, reduces the ability of the solvent to remove the paraffin. Remaining paraffin prevents the dyes from penetrating the tissues, thus giving an uneven appearance.

The simplest way to prevent this from occurring is to change reagents more frequently. Adding a small amount of desiccant pellets about a tablespoon per reagent vessel will also reduce water contamination within solvents. These measures are especially important when using a xylene substitute, as these reagents tend to be far less tolerant of any water contamination than xylene.

When it is properly performed it has the ability to demonstrate a wide range of normal and abnormal cell and tissue components and yet it is a relatively simple stain to carry out on paraffin or frozen sections. Small numbers of slides can be effectively stained manually, while in laboratories that have a high throughput, staining can be performed successfully and consistently by using automated slide stainers.

There are a number of different hematoxylin and eosin formulations in popular use, each with various advantages and disadvantages. Some laboratories prefer to prepare their own solutions whilst others choose ready-to-use commercial products. Hematoxylin precisely stains nuclear components, including heterochromatin and nucleoli, while eosin stains cytoplasmic components including collagen and elastic fibers , muscle fibers and red blood cells.

Following the preparation of a paraffin section, all the elements are infiltrated with and surrounded by paraffin wax which is hydrophobic and impervious to aqueous reagents. The majority of cell and tissue components have no natural color and are not visible.

After thorough de-waxing, the slide is passed through several changes of alcohol to remove the xylene, then thoroughly rinsed in water. The section is now hydrated so that aqueous reagents will readily penetrate the cells and tissue elements. The slide is now stained with a nuclear stain such as Harris hematoxylin, which consists of a dye oxidized hematoxylin or hematein and a mordant or binding agent an aluminum salt in the solution.

Initially this stains the nuclei and some other elements a reddish-purple color. This step converts the hematoxylin to a dark blue color. The section can now be rinsed and checked to see if the nuclei are properly stained, showing adequate contrast and to assess the level of background stain. On most occasions when Harris hematoxylin is employed, a differentiation destaining step is required to remove non-specific background staining and to improve contrast. A weak acid alcohol is used.

After this treatment, blueing and thorough rinsing is again required. The section is now stained with an aqueous or alcoholic solution of eosin depending on personal preference. This colors many nonnuclear elements in different shades of pink. A thin layer of polystyrene mountant is applied, followed by a glass coverslip. If the stain and all the subsequent steps have been properly performed, the slide will reveal all the important microscopic components and be stable for many years.

From patient to pathologist, preparing tissue specimens for histological examination requires care, skill and sound procedures. This guide provides practical advice on best practice techniques and simple ways to avoid common errors. Tips for better routine staining and coverslipping are highlighted in this section.

We hope each step provides a valuable reminder of good histology practice and helps with troubleshooting when unacceptable results do occur. This can produce inconsistent results. This can make it very difficult to determine whether a staining problem is due to poor reagents, an inappropriate protocol or poor fixation.

Agitation, wash and drain times are optimized for all steps during staining. Agitation, wash and drain times are inconsistent. Solvents and reagents rapidly become contaminated. Staining becomes inconsistent. Slide dewaxing is sometimes incomplete, and slides contain patches of residual wax.

This produces unstained, or unevenly stained areas in sections. Solvents and staining reagents are regularly replaced based on the number of slides stained or racks processed. Replacement of solvents and staining reagents is haphazard. They are not replaced until stain quality declines. Hematoxylin solution rapidly becomes contaminated with alcohol and sometimes xylene.

This causes uneven staining. The performance of hematoxylin solutions is carefully monitored. During their working life, hematoxylin solutions are progressively diluted by carry over from slides and racks and are also affected by continuing oxidation. Hematoxylin staining is variable from day-to-day, and no attempt is made to understand why. For example, the staining bath surface area, the extent of aeration during staining, and the ambient temperature can all affect the oxidation rate.

This requirement is influenced by the natural pH of the local tap water. Nuclei that are understained with hematoxylin or over-differentiated and overstained with eosin also appear pink. The pH of the eosin solution is monitored. It is kept close to pH 5. The addition of a couple of drops of acetic acid can be used as a convenient means of lowering pH. No attempt is made to monitor the pH of eosin. When staining intensity falls away the solution is replaced carry over of alkaline tap water can cause the pH of eosin solutions to rise.

Sections are thoroughly dehydrated before being placed in xylene for clearing. Sections are sometimes rushed through alcohol to xylene. Clearing in xylene contaminated with water can result in the presence of tiny water droplets in the tissue that are seen microscopically as opaque areas lacking detail.

The coverslip is always applied before the section has a chance to dry and a high-quality mountant is used. The long-term storage qualities of the mountant must be known because crystals can appear in poor quality mountant,sometimes after a long period months or years. Nucleic acids are acidic , and therefore bind to basic dyes.

Another way of saying this is that nucleic acids are basophilic basic liking. It stains basic or acidophilic structures red or pink. This is also sometimes termed 'eosinophilic'. It is used to stain acidic or basophilic structures a purplish blue.

Haematoxylin is not strictly a basic dye, but it is used with a 'mordant' that makes this stain act as a basic dye. The mordant aluminium salts binds to the tissue, and then haematoxylin binds to the mordant, forming a tissue-mordant-haematoxylin linkage.

This means that the nucleus, and parts of the cytoplasm that contain RNA stain up in one colour purple , and the rest of the cytoplasm stains up a different colour pink. This is a picture of a group of cells lining a duct. The lumen of the duct, and two cells are labelled.