Advanced Visualization Procedures: HISTOCHEMISTRY

Zeliha AHN NEU- Histology and Embryology

 Histochemistry is a method of staining tissue that provides

information about the presence and location of intracellular and extracellular macromolecules
It also reveal where certain chemicals or specific chemical

reactions are taking place within cells or tissues

 Histochemical and cytochemical procedures based on specific binding of a dye with a particular cell or tissue

component the inherent enzymatic activity of a cell component use of an antibody (immunohistochemistry) with a particular cell component, large molecules can be localized by the process of autoradiography.

 Various techniques have been developed to prepare tissues for

study so that they closely resemble their natural, living state. The steps involved are: fixation dehydration and clearing embedding in a suitable medium sectioning into thin slices to permit viewing by transillumination mounting sections onto a surface for ease of handling staining (histochemistry) so that the various tissue and cell components may be differentiated

 The chemical composition of a tissue ready for routine

staining differs from living tissue.

mostly retained in tissue sections

Nucleic acids, proteins and phospholipids (or carbohydrate) are

Small proteins and small nucleic acids (such as tRNA) are generally

lost during the preperation of the tissue.

Neutral lipids are usually dissolve by the organic solvents used in

tissue preperation.

FIXATION
For histochemical analysis, it is essential that the morphology of the

tissues and cells are retained. Fixation refers to treatment of the tissue with chemical agents that stops the alterations of tissue subsequent to death (autopsy material) or after removal from the body (biopsy material) and maintain its normal architecture.
Fixation plays four critical roles in histochemistry : Stabilize cell morphology and tissue architecture Disable proteolytic enzymes Strengthen samples to withstand further processing and staining Protect samples against microbial contamination and decomposition

 Although the most common fixative agents used in light

microscopy are neutral buffered formalin and Bouins fluid, the right fixation method requires optimization based on the application. Methods of fixation:
Chemical Fixation (Perfusion fixation, Immersion fixation) Preserve tissues or cells by cross-link sample proteins, thus

maintaining a lifelike image of the tissue.

Physical Fixation (Drying, Freezing) An alternate approach to prepare samples for staining. This type

of fixation depends on the sample source and the stability of the target antigen.

Drying: Blood smears are air-dried to fix the cells to the slide.

Freezing (frozen sections):

Samples that are too sensitive for chemical fixation can be

embedded in cryoprotective embedding medium and then snapfrozen and stored in liquid nitrogen. Freshly frozen tissue sections are useful for: (1) the demonstration of fats, lipids, and special tissue components; and (2) for rapid preparation of section for histochemistry or microdisection of tissues. Advantages Give better preservation of antigenicity Minimal exposure to fixative Not exposed to the organic solvents Much faster than other forms of fixations. Disadvantages Lack morphological detail

 The frozen tissue is cut in a special refrigerated microtome

called a cryostat and the frozen slices are mounted on a glass slide and stained the same way as other method.

Cryostat

Cutting sections is accomplished in a commercial cryostat microtome at -25C

 Because frozen sections are fast; they're sometimes used for quick

biopsies while a patient is still under anesthesia.

In that case they're stained with Toluidine Blue, and the surgeon

has a pathology "read" within minutes so he can decide what to do. The principal drawback to frozen sections is that they can't be stored: since they're usually not fixed in formaldehyde, they deteriorate rapidly.

Chemical fixation
Perfusion: The circulatory system is cleared of blood and used to

circulate the perfusate uniformly through the body.

Immersion: Samples are immersed in fixative to allow diffusion

through the tissue or cell sample. Both techniques cross-link sample proteins, thus maintaining a lifelike image of the tissue.

 Perfusion fixation: Insert the perfusion needle into the left

ventricle. Perfuse with normal saline to clear the blood from the circulatory system. The eyes of albino rats will turn clear. Perfuse with fixative of choice.

 Immersion fixation: Samples are immersed in fixative

to allow diffusion through the tissue or cell sample

 Although the most common fixative agents used in light

microscopy are neutral buffered formalin and Bouins fluid, no single fixative is ideal for all tissues or samples.
Each fixative procedure must therefore be optimized to balance

adequate fixation without altering the cellular detail of the tissue The most common fixative agents used in light microscopy Formaldehyde: 10% neutral buffered formalin solution Bouins fixative (Picric acid fixative) Zenkers solution (Acetic acid fixative) Precipitating solutions (ethanol, acetone or methanol)

Dehydration & Clearing

Because a large fraction of tissue is composed of water, a graded

series of alcohol baths (50%, %70, %95%, 100%) are used to remove the water (Dehydration = freeing from the water).
Once absolute ethanol is achieved, the tissue is then treated with

xylene, a chemical that is miscible with melted paraffin. This process is known as clearing, because the opaque tissue becomes transparent in xylene.

Embedding
In order to distinguish the overlapping cells in a tissue and the

extracellular matrix from one another, the histologists must embed the tissues in a proper medium and then slice them into thin sections. For light microscopy, the usual embedding medium is paraffin.

 The tissue is placed in a suitable container of melted paraffin until it

is completely infiltrated. Once the tissue is infiltrated with paraffin, it is placed into a small receptacle, covered with melted paraffin, and allowed to harden, forming a paraffin block containing the tissue.

Paraffin Blocks

Sectioning & Mounting

Embedded tissue is removed from its mold, trimmed, mounted for

sectioning. The cutting is performed using an instrument called microtome, a machine equipped with a blade and an arm. For light microscopy, the thickness of each section is about 5-10 m.
Paraffin sections are then mounted (placed) on glass slides and then

stained by water-soluble stains that permit differentiation of the various cellular components.

a microtome

 The sections for conventional light microscopy, cut by stainless

steel blades, are mounted on adhesive-coated glass slides.

Because many tissues constituents have apprx. the same optical

densities, they must be stained for light microscopy, usually with water-soluble stains.

 Therefore, the paraffin must be first removed from the section,

after which the tissue is rehydrated and stained.

After staining, the section is again dehydrated so that the coveslip

may be permanently affixed by the use of a suitable mounting medium.

The coverslip not only protects the tissue from damage but also is

necessary for viewing the section with microscope.

Harvesting the tissue

Coverslipping
dehydration

A piece of tissue
cutting the tissue into small pieces

staining

Rehydration (graded series of ethanol)

Remove paraffin

PARAFFIN TECHNIQUE

fixation

Mounting tissue on a slide

Dehydration (graded series of ethanol)

Embedding

Staining & Histochemistry

Various types of stains have been developed for visualization of the

many components of cells and tissues. These procedures take advantage of the intra- and extra- cellular chemistry of the tissues. Stains may be grouped into three classes. Stains that differentiate between acidic and basic component of the cell Specialized stains that differentiate the fibrous components of the extracellular matrix Metallic salts that precipitate on tissues, forming metal deposits on them

 An acidic dye, carries a net negative charge on its colored

portion and is described by the general formula [Na+dye-].

A basic dye carries a net positive charge on its colored

portion and is described by the general formula [dye+ Cl-].

 Basic dyes+ react with anionic components- of cells and tissue

Anionic components: phosphate groups of nucleic acids, sulphate

groups of GAGs, the carboxyl groups of proteins. The ability of such anionic groups to react with a basic dye is called basophilia.
Acidic dyes- react with cationic components+ of cells and

tissue
Cationic components: amino groups of proteins The ability of such cationic groups to react with a acidic dye is

called acidophilia.

 Basophilic substances include:

Heterochromatin and nucleoli of the nucleus Cytoplasmic components such as the ergastoplasm, Nissl Extracellular materials such as complex carbohydrates of the

matrix of cartilage
Acidophilic substances include:
Most cytoplasmic filaments Most intracellular membranous components

Most extracellular fibers

Basic Dyes+ (Anionic Dye-): Hematoxylin Azure Methylene blue Toluidine blue Thionine Basic Fuchsin Safranin

Acidic Dyes- (Cationic Dye+): Eosin Aniline blue

Picric acid Fast green Orange G Light Green Acid Fuchsin

Congo red

Common Histochemical Stains

STAIN TYPE COLOR SPECIFICALY STAINS

Hematoxylin

Basic dye

Blue

Nucleic acids, cytoplasmic and extracellular anionic components (RER, proteoglycan) Cellular and extracellular cationic components (e.g. collagen, muscle proteins)

Eosin Orange G Picric acid Aniline blue Toluidine blue Methylene blue Azures

Acidic dye

Pink-Red

Basic dye (metachromatic stain)

Blue

Cellular & EC anions, metachromatic (high density polyanions),purple e.g. mast cells granules, basophil granules, cartilage ECM)

Alcian blue

Basic dye

Blue-green

High density GAGs (mucinogen)

STAIN

TYPE

NUCLEUS

SPECIFICALY STAINS

Massons trichrome

Connective tissue

Black Red Green -Blue Red Blue Black/ blue

Nuclei Cytoplasm, muscle Cartilage, collagen Muscle fiber Cartilage, Bone matrix Elastin fibers Muscle-yellow Cytoplasm-yellow Collagen-red

Mallorys Trichrome

Connective tissue

Verhoeff van Gieson

Elastin fibers

Orcein stain

Elastin fibers

Red (Brick red)

Elastin fiber Smooth muscle- light blue

STAIN

TYPE

COLOR

SPECIFICALY STAINS

Wrigt and Giemsa Blood stains

Bluish/purple

Neutrophil gr.-pink Eosinophil gr.-bright red Basophil gr.-deep purple Platelet gr-red/purple
Lipid

Sudan black Oil Red O Sudan III

Lipid stains

Black Red Black

STAIN

TYPE

COLOR

SPECIFICALY STAINS

PAS (periodic acid-Schiff) Silver

Glycols

Magenta

Basement memb., carbohydrates, glycogen Golgi apparatus, reticular fibers, neurofibrils (argyrophilia; affinity for silver)

Metal

Black

HEMATOXYLIN & EOSIN (H&E)

Stains anionic components: Nucleic acid, DNA, RNA

Hematoxylin

Eeosin components: Stains cationic

H&E

cytoplasmic proteins, certain fibers- collagen

HEMATOXYLIN & EOSIN (H&E)

cytoplasm nucleus

HEMATOXYLIN & EOSIN (H&E)

HEMATOXYLIN & EOSIN (H&E)

HEMATOXYLIN & EOSIN (H&E)

HEMATOXYLIN & EOSIN (H&E)

Alcian Blue

Metachromasia
Certain basic dyes react with tissue components that shift their

normal color from blue to red or purple; this absorbance change is called metachromasia.
The underlying mechanism for metachromasia is presence of

polyanions within the tissue. When these tissues are stained with a concentrated basic dye solution, such as toluidine blue, the dye molecules form dimeric and polymeric aggregates. These aggregates differ in colour from their individual molecules.

Metachromasia
Cell and tissues that have ionized sulphate and phosphate

groups:
ground substance of cartilage
Heparin-containing granules of mast cells Rough endoplasmic reticulum of plasma cells

exhibit metachromasia. Therefore, toluidine blue, methylene blue will appear purple to red when it stains with these components.

Metachromasia
Tissue / cell component that

stains purple with these basic dyes is said to be metachromatic.

The dye is said to exhibit

metachromasia.

Mast cells

Metachromatic Mast cells

Metachromatic Cartilage ECM

Mallorys Trichrome

Nucleus: bright red

Cytoplasm: Pale pink

Ground substance (collagen and reticulum fibers): blue

Mallory s Trichrome

Mallory s Trichrome

Massons trichrome
Cytoplasm: red / pink

Collagen: green

A three-colour staining protocol. Suited for distinguishing cells from surrounding connective tissue

Muscle: red / pink

Collagen: green

Nuclei: blue/black

Massons trichrome

Massons trichrome

Verhoeff s Van-Gieson
Muscle: yellow

Collagen bundles: pink/red

Elastic fibers: black

This stain is used to demonstrate elastic fibers in all tissue types.

Verhoeffs Van-Gieson

Orcein stain

a reddish-brown dye
Distinguish

elastin fibers

Silver staining

Use of silver to stain fibers

(reticular and nerve fibres)

Silver staining

GOLGI COMPLEXES

Silver staining

Silver staining

Wright and Giemsa stains

Wright and Giemsa stains

are used to study blood cell morphology.

cytoplasm: orange to pink

nucleus: blue to purple.

Lipids with H&E stain

Frozen section, lipid stain

Frozen section, lipid stain

OIL RED O

SUDAN BLACK

PAS (Periodic Acid-Schiff) Reaction

Stains

carbohydrates carbohydrate-rich macromolecules.

and

It

is used to demonstrate glycogen and mucus in various cells, the basement membrane that underlies epithelia and reticular fibers in connective tissue.

PAS (Periodic Acid-Schiff) Reaction

Feulgen reaction:
The Feulgen reaction takes advantage of the ability of hydrochloric

acid to convert DNA to an aldehyde. Again, the newly formed aldehyde group react with the Schiff reagent which is specific for aldehydes. The reaction of Schiff reagent with DNA is stoichiometric, meaning that the product of this reaction measurable and proportional to the amount of DNA. It can be used to quantify the amount of DNA in the nucleus of a cell. RNA does not stain with the Schiff reagent because it lacks deoxyribose.

Enzyme Histochemistry (Enzyme Digestion)

Histochemical methods are also used to identify and localize

enzymes in cells and tissues. Special care must be taken in fixation to preserve the enzyme activity The reaction product of the enzyme activity, rather than the enzyme itself, is visualized.

 Intracellular material that stains with the PAS reaction may be

identified as glycogen by pretreatment of sections with diastase or amylase. Abolition of the staining after these treatments positively identifies the stained material as glycogen.
Similarly, pretreatment of tissue sections with DNAase will abolish

the Feulgen staining in those sections, and treatment of sections of protein secretory epithelia with RNAse will abolish the staining of the ergastoplasm with basic dyes.

Detectable enzymes: Acid hydrolase Alkaline phosphatase Adenosine triphosphatases (ATPases) Succinate dehydrogenase Peroxidase Various esterases Many respiratory enzymes

acetylcholine esterase (ACHE)-positive fibres (brown) in the lamina propria mucosae

Demonstration of Alkaline phosphatase by Gomori method

 Electron micrograph of a rat kidney cell. The dark precipitate within these structures is lead phosphate precipitated on places where acid phosphatase was present. x25,000. (E. Katchburian.)

Some vital stains (cell viability)

Propidium iodide (PI):

A fluorescent dye, stains DNA . The principle: apoptotic cells are characterized by DNA fragmentation and,

consequently, loss of nuclear DNA content. Use of PI, that is capable of binding and labeling DNA makes it possible to evaluate the cellular DNA content. Trypan Blue : Trypan Blue Stain is a stain used to distinguish viable from nonviable cells. Nonviable cells will absorb the dye and appear blue, while viable cells will exclude the dye. Applied to living cells in culture medium

Brightfield image of Jurkat cells.

Fluorescent image indicating PI stained dead Jurkat cells.

ELECTRON MICROSCOPE TECHNIQUE

Fixation (gluteraldehyde) Dehydration (ethanol) Clearing, (propilene oxide) Embedding, (plastic resin) Plastic blocks Sectioning with ultramicrotome Staining for LM, (methylene blue) Trimming Sectioning Staining with heavy metals (osmium tetraoxide)

TEM (TRANSMISSION ELECTRON MICROSCOPY

TEM (TRANSMISSION ELECTRON MICROSCOPY

TEM (TRANSMISSION ELECTRON MICROSCOPY

SEM (SCANNING ELECTRON MICROSCOPY