Hematoxylin is a clear-contrast differentiator that is used in a variety of applications, including staining cells and other materials. It is also used as a stabilizing agent for iron mordants in a variety of processes. Among other uses, it is also a counterstaining agent and is used in nuclear fast red and methyl green staining.
Staining cells
The Hematoxylin and Eosin (H&E) stain is a popular histological stain for the histologic examination of human tissue. It has a broad range of features that are used to distinguish various cellular elements. A proper H&E stain allows pathologists to differentiate between benign and malignant cells.
The hematoxylin and eosin stain provides detailed and precise information about the structure of the specimen. The blue-purple staining of the hematoxylin gives a strong contrast to the cytoplasm, which is pinkish. Depending on the type of tissue, the intensity of the staining will vary.
The hematoxylin produces a blue-purple color that stains the nuclei of the cell. This color is caused by oxidation reactions with nuclear histones. In addition, the hematoxylin and alumni complex binds to phosphate groups of DNA.
Eosin, on the other hand, adds a pink tone to the cytoplasm and connective tissue. Various cellular organelles are eosinophilic. Examples of eosinophilic structures include the nucleus, the extracellular matrix, and the ribosomes.
Although the hematoxylin and eosin (H&E) stain remain the gold standard for histology, there are various regressive and progressive hematoxylins available. Regressive hematoxylins are used to remove excess hematoxylin, while progressive hematoxylins are used for differentiation.
To determine the appropriate staining technique, you can use a guide. For example, the Richard-Allan Scientific Signature Series Hematoxylin 7212 contains a special formula that produces a strong and consistent staining intensity.
A well-fixed tissue section shows considerable intranuclear detail. This information can help pathologists to determine the nature and progression of the disease. With the proper staining technique, a lab technician can also differentiate the cellular components on the tissue section.
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Counterstaining hematoxylin
In nuclear staining, hematoxylin is the most common counterstain. It is a naturally occurring substance that undergoes chemical oxidation to generate the oxidation product known as blue hematin, which is used to dye mammalian cells. The resulting dye is then oxidized to aluminum ions to form an active metal-dye complex. This reaction results in the formation of tetrabro-mofluorescein, a fluorescent dye that can be used to identify individual molecules.
Hematoxylin staining is typically performed with a mordant. There are many different types of mordants. Common ones include lead, iron salts, and mercuric reagents.
Various dyes are also available for nuclear staining. They may be chromogenic, fluorescent, or dyes that stain specific chemical molecules. Depending on the experimental design, the dye will stain a specific cellular compartment or cellular structure.
A hematoxylin and eosin stain, or H&E stain, is one of the most important histology stains. It allows the staining of red blood cells and smooth muscle, as well as the localization of nuclei.
Eosin is a strong anionic dye, and its affinity for protein structures makes it an effective counterstain. Its guanidino group of arginine binds to most protein structures, especially the side chain of lysine. Unlike hematoxylin, which stains nuclear materials, eosin stains extracellular matrix and cytoplasm pink.
Hematoxylin and eosin is an excellent histology stain for the nucleus, but it is also useful for visualizing other cellular structures. For this reason, it is frequently used in cytology laboratories.
When it comes to nuclear staining, there are two main types of hematoxylin. These are progressive and regressive hematoxylins. Progressive hematoxylins are a less strong form of hematoxylin that is used to differentiate tissue. Regressive hematoxylins are stronger forms of hematoxylin used to remove excess hematoxylin.
Stabilizing iron mordants
Stabilizing iron mordants for hematoxylin clear-contrast-differentiator formulations is an issue that continues to occupy researchers and manufacturers.
The main reason for this is that hematoxylin is expensive because of the presence of nuclear stains.
Among the many oxidants that can be used to convert hematoxylin to hematein are hydroquinone, sodium periodate salts, and iodate salts.
These compounds have been known to increase the staining kinetics and reduce the likelihood of precipitation.
One proposed method of stabilizing hematoxylin is by modifying the molar ratio of sulfate to chloride.
Chloride is a more common anion than sulfate, and it dissolves more readily. However, the amount of chloride added would be limited by the pH of the solution.
In addition, a substoichiometric concentration of oxidant may be used to reduce the amount of hemalum formed. This approach can prevent the overoxidation of ineffective products.
Although the molar ratio of sulfate and chloride is not required, a substoichiometric molar ratio of sulfate-containing salts such as mercuric oxide or potassium aluminum sulfate can increase the oxidant concentration.
The molar ratio of chloride-containing salts can be modified to match the CIVSC2" molar ratio of hematoxylin.
Another approach to stabilize hematoxylin solutions is by adding an alkaline liquid. An alkaline solution can be used to neutralize the acidic pH and accelerate the blueing process. A liquid can be placed at 600C for 30 minutes. If the water is too acidic, a tap water substitute can be used.
Using a combination of aluminum sulfate and aluminum chloride as the sulfate-containing salts can produce solutions that stain as quickly as those based on sulfate alone. It was found that removing the S04 2" from ANS-Cl resulted in inferior kinetics.
Nuclear fast red
Hematoxylin is the most common nuclear stain in histopathology. It is also used in immunohistochemistry for the identification of specific antigens. There are several types of hematoxylin and different mordants.
Various iron salts are commonly used as mordants for hematoxylin. Iron hematoxylin produces a black color that stains nuclei. Lead is another common mordant. Aluminum and tungsten salts can be used, too.
The type of mordant used in hematoxylin-eosin stains determines the contrast between the nuclei and eosin. In general, hematoxylin-eosin-stained sections produce grayscale values of seven or eight. However, there are variations in the color intensity depending on the type of hematoxylin used.
Regressive hematoxylin is used to remove excess hematoxylin. This stain is a convenient way to stain cells. Progressive hematoxylins are more convenient than regressive hematoxylins. They allow for fine control over the density and color of stained nuclei.
The grayscale value of the nuclei varies between 7 and 9. HE colors are higher in grayscale values than reds, blues, and greens. HE stains provide excellent color contrast. Figures 9-17 illustrate HE colors.
Phloxine is a counterstain for hematoxylin. Phloxine reduces the contrast between hematoxylin-stained structures and eosin-stained ones. Compared to hematoxylin-eosin, phloxine stains more magenta.
Another regressive hematoxylin used in immunohistochemistry is Mayer's. Mayer's hematoxylin solution uses aluminum alum. Unlike Harris's hematoxylin, it is easier to maintain a mid-gray value with this counterstain.
Depending on the type of hematoxylin, grayscale values can range from seven to nine. Optimal grayscale values are essential for effective photomicrography and histopathology. Using the correct hematoxylin-eosin combination allows for maximum grayscale contrast.
Choosing the correct hematoxylin-eosin counterstain depends on the purpose for which the tissue is being examined. Ultimately, you must decide the level of intensity and detail you want to see.
Methyl green
Hematoxylin is a strong nuclear dye and is used to stain nuclei. It is extracted from the logwood tree and is then combined with aluminum. The combination gives a light blue color.
Hematoxylin is used in a wide variety of immunohistochemical stains. In particular, methyl green is used for detecting DNA. Another clear-contrast-differentiator is a combination of methyl green and pyronin Y. This staining method has been employed in histochemical staining and PCR amplification.
Using a variety of hematoxylins allows you to achieve a greater range of grayscale values. Some of these hematoxylins, such as Gill's and Mayer's, have a high density of staining. Other hematoxylins have a lower density of staining, such as alum hematoxylin.
Although hematoxylins can be stained with a variety of mordants, the maximum density of staining in an HE stain depends on the type of mordant. Optimal staining results in grayscale values of seven to nine. However, if the background is too dense, the result can be muddied. To minimize this effect, phloxine staining can be applied.
When using hematoxylin, it's important to understand the difference between a weak blue and an intermediate color.
An intermediate color is a blue-green color which is not quite as dense as a weak blue.
A weak blue is a color that is nearly identical in intensity to an intermediate color.
Despite its apparent advantages, weak blue is not as color-neutral as weak red, green, or yellow.
Choosing the right hematoxylin is a daunting task. You'll want to consider the type of hematoxylin, the types of dyes, and the underlying technique.