HISTOLOGY STAINING

Hematoxylin and Eosin Stain

The H&E stain is the stain routinely performed in Histology laboratories, but it is just as special as the so called “special stains”. It is a special stain for the nucleus. Hematoxylin, of one formulation or another, is used as a nuclear stain and Eosin is used, either alone or in combination with other plasma stains, as the counterstain. The stain theory is based on the attraction of oppositely charged tissue and dye molecules.

 

 

Nuclear Stains

 

Hematoxylin is extracted from logwood, otherwise known as campeachy wood. It is not a dye.  The oxidized product of hematoxylin, hematein (pronounced with four syllables), is a weak anionic dye with very little affinity for tissue. Hematein becomes a strong dye that will only exhibit an affinity for nuclei if it is combined with a metallic mordant, most commonly aluminum or iron and occasionally tungsten. The mordant-dye combination is called a lake. The metal-hematein lakes have traditionally been considered basic, or cationic, dyes, but there is debate in the literature. Most routine stains are done using aluminum, sometimes referred to as alum, hematoxylins such as Harris, Gill and Mayer. Iron, or ferric, hematoxylins, such as Weigert, are used as nuclear stains in many special techniques because they resist decolorization in acidic staining solutions.

 

Hematoxylin can be used as either a progressive or a regressive stain. In progressive staining, the reaction is stopped once the desired staining intensity is achieved. In regressive staining, the tissue is overstained and then differentiated in a dilute aqueous or alcoholic hydrochloric acid solution to achieve the desired results.  The color shifts from blue/purple to a salmon pink/red.  After differentiation, the sections are rinsed and then placed in a dilute alkaline “bluing” solution to reestablish the insoluble (blue colored) dye lake.  A thorough rinse is necessary after this step to avoid adversely influencing the counter staining step.

 

The nucleus reflects the reproductive potential of the cell.  The size and staining intensity (chromasia) of the nucleus is a critical factor in the evaluation of the cell.  Correct Hematoxylin staining shows crisp staining of the nuclear chromatin, demonstrating well delineated nuclear membranes and sharply stained condensed chromatin against an unstained nucleoplasm. 

 

Harris Hematoxylin is one of the more common formulations used in the H&E stain.  It can be used progressively but is usually used regressively. The original formula used mercuric oxide as the oxidizing agent but, due to its toxicity, modern modifications substitute sodium or potassium iodate. Solutions used at a low pH provide very selective nuclear staining. Most Harris hematoxylins form a metallic precipitant upon standing, so they should be filtered before use.

 

Harris Hematoxylin ( CAT# SL90) is a modified formula that is non-acidified and mercury free.  Potassium iodate is used to oxidize the solution.  This ready to use staining solution produces exceptional nuclear detail when used in either progressive or regressive protocols.  It comes in 16 ounce or 1 gallon bottles and 2.5 gallon cubitainers.

 

Gill’s Hematoxylin uses ethylene glycol as the solvent for the hematoxylin in order to prevent the formation of surface precipitants and assist with diffusion and penetration.  Sodium iodate is used as the oxidizer and aluminum as the mordant.  It is usually used progressively and has gained wide acceptance in histology and cytology staining procedures.  One of its distinguishing characteristics is that it stains goblet cells.

 

Gill 1 (CAT# SL97), Gill 2 (CAT# SL94) and Gill 3 (CAT# SL95) Hematoxylins have gained wide acceptance in histology and cytology.  They contain the same constituents but progress in strength and staining intensity, making it possible to obtain similar results when using any of the three solutions simply by altering the exposure times.  Each formula is available in 16 ounce or 1 gallon bottles and 2.5 gallon cubitainers.

 

Bluing Reagent (CAT# SL99), or Scott’s solution, is a closely controlled pH balanced solution that provides consistent and rapid bluing of hematoxylin stained nuclei.  It is available in 16 ounce or 1 gallon bottles and 2.5 gallon cubitainers.

 

 

Cytoplasmic Stains

 

The plasma stains are most frequently anionic (negatively charged) dyes with an affinity for cationic positively charged) tissue groups.  Because of the terminal amino (-NH₂) and carboxyl (-COOH) groups commonly found on proteins and some side chains of amino acids, proteins may be either positively or negatively charged (amphoteric).  The charge is dependent on the pH.  When the pH of the stain solution is below pH 6.0, the isoelectric point (IEP) of proteins, the proteins are generally basic or positively charged.

 

Eosin is a widely used counterstain that provides its maximum uptake to the basic cytoplasmic proteins when used at pH 4.6 to 5.0.  If the pH is too low, the eosin will convert to free acid, dominate the solution and yield a muddy stain.  If it is too high, the net charge on the protein will become negative and it will have no affinity for the dye. Used properly it will provide at least three shades of pink, showing different intensities on erythrocytes, collagen and the cytoplasm of muscle or epithelial cells. The single dye can stain different shades because the dye molecules can aggregate in a concentrated solution or on the tissue section if the charged sites are close together. The aggregation of eosin molecules causes a rearrangement of chemical bonds which produces slightly different colors of the dye.

 

1% Eosin Y (CAT# SL98) is a stable, precipitant free solution.  The pH is adjusted within a range of 4.0 to 5.0 in order to provide varying staining intensities on different tissue constituents.  It is available in 16 ounce, 32 ounce or 1 gallon bottles and 2.5 gallon cubitainers.

 

Treosin™ is a proprietary mixture that may be substituted for eosin in any staining method.  It is a slightly acidified combination of Eosin Y and Orange G with the addition of acid fuchsin. It is excellent for frozen sections or for intense histologic staining. It provides greater intensity and more vibrant colors than with eosin staining, with colors ranging from pink, to red-orange to bright red. The stain also differentiates between collagen and smooth muscle so it may reduce the need for trichrome stains.

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H&E Staining