Haematoxylin alone is not technically a dye, and will not directly stain tissues. The mordant used is typically a metal cation, such as aluminium. Haematoxylin in complex with aluminium salts is cationic and acts as a basic dye. It is positively charged and can react with negatively charged, basophilic cell components, such as nucleic acids in the nucleus. These stain blue as a result.
Eosin is anionic and acts as an acidic dye. It is negatively charged and can react with positively charged, acidophilic components in the tissue, such as amino groups in proteins in the cytoplasm. These stain pink as a result. What are your tips for tissue section staining? Has this helped you? Then please share with your network. Why Haematoxylin is used first and not Eosin? The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features.
Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. Mercuric oxide was used as the oxidant of choice in Harris formations for many years but is no longer used due to environmental issues concerning mercury.
Today, mercuric oxide is replaced with sodium iodate in modified Harris formulations. In contrast to hematoxylin which has been well characterized structurally, the molecular structure of hematein has been debated in recent years. The traditional structure of hematein which includes a quinoid ring is depicted in Figure 1. Regardless of the exact structure of hematein, we can assume that hematein is structurally similar to hematoxylin based upon NMR analysis and similarities in molecular weight.
In addition, the oxidation to hematein is necessary in order to bind metal ions such as aluminum. In addition to the oxidants and aluminum salts described above, hematoxylin solutions often contain other additives or agents. Modifications to the solvent are made by the addition of polyhydroxy alcohols such as glycerin or more commonly ethylene glycol.
Initially it was proposed that the polyhydroxy alcohols acted as stabilizers to reduce the over oxidation of hematein. The inclusion of ethylene glycol in the solution reduces the precipitation or deposition of these products and thus increase the stability or shelf life of the hematoxylin solution. Various acids are sometimes added to hematoxylin solutions to reduce the pH. Acetic acid is added to Gill type hematoxylins while citric acid is found in Mayers.
The addition of acids to hematoxylin solutions increases the selectivity of the stain for nuclei by reducing nonspecific background staining. A number of varied theories have been proposed to account for the staining of nuclei by hematein-Al complexes. Nuclear molecules that have been proposed to bind hematein-Al complexes include basic positively charged proteins such as histones as well as deoxyribonucleic acid DNA.
Why are the nuclei in the image shown above different sizes? Are they really different sizes, or is there an alternative explanation? Hint, think about what happened when the sections were cut, and about how big the cells are. What kinds of histological stains are there? Basophilic and acidophilic staining.
Can you spot the nuclei? Other kinds of stains There are many other kinds of stains each of which stain tissues in characteristic ways. Questions for you to think about.
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