Histological Stains 1

7/29/2019 Histological Stains 1

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Histological Stains

Staining Reactions

Staining reactions have both physical and

chemical characteristics. The mechanisms

involved in staining include the following:

The dye may actually be dissolved in the stained

substance. Most fat staining is accomplished in

this fashion. A dye may be absorbed on the

surface of a structure, or dyes may be

precipitated within the structure, simply because

environmental factors (pH, ionic strength,

temperature, etc.) favor absorption or

precipitation. Most staining reactions involve a

chemical union between dye and stained

substance through salt linkages, hydrogen bonds,or others. Staining with these dyes results in a

predictable color pattern based in part on the

acid base characteristics of the tissue. However,

color and color distribution are not absolutely

reliable for discrimination between tissue

components. Color will vary not only with specific

stains used, but also with the conditions that exist

during preparation of the slide. These include

everything from the initial fixing solution to the

ionic strength of the staining solution and the

differentiating solvents utilized after staining.

Acid and Basic Dyes

Most histologic dyes are classified either as acid or

as basic dyes. An acid dye exists as an anion

(negatively charged) in solution, while a basic

dye exists as a cation (positive charge). For

instance, in the hematoxylin-eosin stain (H&E), the

hematoxylin-metal complex acts as a basic dye.

The eosin acts as an acid dye.

Any substance that is stained by the basic dye is

considered to be basophilic; it carries acid groupswhich bind the basic dye through salt linkages.

When using hematoxylin, basophilic structures in

the tissue appear blue (or purple or brown; this

varies according to the stain that is being used). A

substance that is stained by an acid dye is

referred to as acidophilic; it carries basic groups

which bind the acid dye. With eosin, acidophilic

structures appear in various shades of pink. Since

eosin is a widely used acid dye, acidophilic

substances are frequently referred to as

eosinophilic.

Trichrome Stains

In the trichrome stains, which commonly employ

more than one acid dye, use is made of dye

competition. For instance, acid fuchsin and picric

acid are used in Van Gieson's trichrome stain. In

the picric acid-fuchsin mixture, the small picric

acid molecule reaches and stains the available

sites in muscle before the larger fuchsin molecules

can enter. Used by itself, acid fuchsin has no

difficulty in staining muscle.

Neutral Stains

These are compounds of an acid dye and basic

dye. For instance, aqueous solutions of acid

fuchsin may be neutralized by addition of

aqueous methyl green. The resulting neutral

product is water insoluble, but may be kept in

solution by the presence of excess amounts of

either component. The tissue stained with such a

solution may show affinity for the acid dye, the

basic dye, and for the whole compound. Some

blood stains are "neutral stains." Wright's Stain, for

instance, is formed by the combination of partiallyoxidized and demethylated methylene blue and

eosin. Such a stain can be used to differentiate

between blood cells that contain acidic, basic,

and neutral granules.
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Characteristics of Commonly used Stains

Hematoxyl and Eosin (H&E)

This is a good general stain and is widely used.

Most of your slides are stained with H&E. A

hematoxylin-metal complex acts a as a basic

dye, staining nucleic acids in the nucleus and the

cytoplasm blue, brown, or black. Eosin is an acidaniline dye which stains the more basic proteins

within cells (cytoplasm) and in extracellular

spaces (collagen) pink to red. Cartilage and

mucus may stain light blue. Example: slide 42c.

Masson Trichrome Stain

A staining sequence involving iron hematoxylin,

acid fuchsin, and light green. It is a good stain for

distinguishing cellular from extracellular

components. Collagen fibers stain an intensegreen. Black or brown nuclei; mucus and ground

substances take on varying shades of green.

Cytoplasm stains red. Elastic fibrils, red blood cells

and nucleoli stain pink. Example: slide 56-l.

Aldehyde Fuchsin

Stains elastic fibers purple to black. Can be

counter-stained with a dye of contrasting color,

such as metanil yellow. Example: slide 42d.

Verhoeff's Hematoxylin

Another variant of the versatile hematoxylin stains.

This method stains elastic fibers black in addition

to nuclei. Example: slide 71d.

Reticular Fiber Stain - Weigert

Reticular fibers are impregnated with a silver salt

and appear as sharp black. Collagenous fibers

usually stain purple. This stain can be used with a

counterstain (such as slide 21c) or without, if the

silver stain turned out very dark (such as slide 22b).

Wright's/Giemsa Stain

This and similar stains for blood and bone marrow

smears are mixtures of basic (methylene blue

derivatives) and acid dyes (usually eosin).

According to the number of acid and basic

groups present, cell components take up the dyes
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from the mixture in various proportions. Example:

slide 17b. Some blood stains use acid and basic

dyes in separate dye baths.

Metachromatic Stain

Certain basic dyes, such as toluidine blue, stain

nucleic acids blue (the orthochromatic color), but

sulfated polysaccharides purple (themetachromatic color). When dye molecules

bound to sulfate groups are stacked closely

together, the dye experiences a color shift from

blue to purple. Thus, a metachromatic reaction

often indicates the presence of numerous closely

packed sulfate groups.

Plastic Sections Stained with Toluidine Blue or with

H&E

Plastic embedded tissues can be cut as thin as 0.1

m with a glass knife. These sections are thenstained with toluidine blue in an alkaline solution.

Almost all tissue components are stained more or

less deeply (usually a bluish-purple) and structural

detail is very sharp. For the knowledgeable

observer, this type of preparation may be very

informative. Example: slide 56c.

Histochemical Techniques

In contrast to the staining techniques described

above, the mechanism of a histochemicaltechnique is usually well understood. If properly

controlled, the technique is chemically specific,

highly reproducible and capable of providing

information that can then be quantitated. The

following two methods make use of leuko-fuchsin,

also known as the Schiff's Reagent.

Periodic Acid Schiff (PAS)

Adjacent hydroxyl groups (1, 2 glycols) or amino

and hydroxyl groups are oxidized to aldehyde

groups with periodic acid. Schiff's Reagent then

produces a red or magenta addition product with

the aldehyde groups and this technique identifiesa number of polysaccharides and carbohydrate-

containing compounds. The slide may also be

counter stained with hematoxylin. Example: slide

56b. Feulgen Reaction: Mild hydrolysis with HCl

frees the aldehyde group of deoxyribose, which is

then reacted with the Schiff's reagent. This

reaction is highly specific for DNA and may also

be used with a counter stain for the cytoplasm.

Enzyme-histochemical Techniques

These techniques localize various enzymes within

cells and tissues by making use of the enzyme

activity itself. The enzyme is made to react with a

specific substrate. The product of this reaction

may itself be visible in the microscope and thus

demonstrate the presence of the enzyme at a

specific location, or the reaction product is

subsequently reacted to form a visible secondary

reaction product. Example: Reaction for acid

phosphatase. 1) The tissue is gently fixed in order

to preserve acid phosphatase activity. 2) Sections

are incubated in a medium consisting of beta-

glycerophosphate (the specific substrate), lead

nitrate ("the capturing agent" for the secondary

reaction), buffer at pH 5 (the pH for optimal

activity of the acid phosphatase). During

incubation, the acid phosphatase splits

phosphate groups off the beta-

glycerophosphate.

The phosphate groups immediately react with the

lead ions to form insoluble lead phosphate, which

precipitates at the location of the enzyme. Lead

phosphate is visible in the electron microscope or

can be made visible in the light microscope with

a further reaction. This type of reaction can be

modified to demonstrate a wide variety of

enzymes.
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Immunohistochemical Techniques

These techniques are well described in the text.

They are the most specific and sensitive methods

available for identifying specific substances

(antigens) within cells and tissues. They utilize

antibodies that are made against specific

antigens and enable us to localize specific

substances within cells and tissues.

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Histological Stains 1