HISTOCHEMICAL

and

IMMUNO-HISTOCHEMICAL
TECHNIQUES

HISTOCHEMICAL TECHNIQUES
Studies various tissue constituents through chemical reactions that enable to permit microscopic localization of a specific tissue substance. Histochemical identification:

carbohydrates
cystine and cysteine proteins

Histochemical staining technique

Tissue constituent

Principle
To oxidize the polysaccharides and mucin to liberate the aldehyde through the use of schiffs regent

Reagents use

Resulting color
Nuclei blue black/blue Positive subs. red/magenta green

PAS

carbohydrates proteins Cystine and cysteine

Alkaline fast green Peracetic acidalcian blue

Schiffs reagent

To stain ionized basic group such as protamines and histones To oxides cystine and cysteine to form cysteic acid which stained blue-green by a basic dye

Fast green Trichloroacetic acid

Peracetic acid Alcian blue

Blue-green

Pas positive

Alkaline fast green

Histochemical staining technique

Tissue constituent

Principle

Reagents use
Schiffs reagent

Resulting color
DNA RED PURPLE Cytoplasm GREEN

Nucleic acid DNA

Feulgen technique

to liberate aldehyde groups from the containing sugar of the nucleic acid. It will then stained the liberated aldehyde group which is acted upon by the schiffs reagent. Anionic phosphate group of the DNA is highly selective and is able to bind to the stained giving the DNA a green color, while Pyronin will be acted to RNA giving it a pinkish color.

RNA AND DNA

Methyl greenpyronin mtd

Methy green pyronin

DNA (chromatin) GREEN or BLUE GREEN RNA (nucleoli) ROSE RED; GRANULES (DARK RED ROSE) Plasma cell cytoplasm PURPLE

Feulgen DNA

Methyl green pyronin

Enyzme histochemistry
enzyme mtd Gomori lead principle
Phosphates reacts on Ca ions to form Ca Phosphate which is then treated with cobalt nitrate it will produce Co phoshates which gives a black precipitate Adenosine-5phosphate is hydrolyzed to phosphate ions in the presence of Mg. it is then precipitated by lead ions to produce lead phosphate Uses a substrate to form phosphate ions which will then precipitated by lead to form lead phosphates

Reagent use
Sodium glycerophosphate

Resulting color Acid Phosphatas e activity black Nuclei green

Acid phosphatase

5nucleotidas e

Lead

AMMONIUM SULFIDE SUBSTRATE

BROWN

Adenosine triPHOSPH ATASE

Metal precipitation

Lead Ammonium sulfate

Brownish precipitate

Enyzme histochemistry
enzyme mtd principle Reagent use Resulting color
Esterase blue Nuclei - red

Indoxyl acetate mtd

non-specific esterase
Alpha napththy acetate

Uses a substrate that will be then produce bromo indoxyl that will be then oxidized to an insoluble dye Uses substrate that releases napthol during enz hydrolysis and will form an insoluble dye at the site of the enzyme activity Demonstrated thru the oxidation of TNBT

bromo-indoxyl acetate (substrate)

a-napththyl acetate (substrate)

Diazonium salt

Esterase reddish brown Nuclei green

Monoamine oxidase BLUISH BLACK

monoamine oxidase

Tetrazolium mtd

tetra nitro-blue tetrazolium (TNBT)

LEAD METHOD OF 5-NUCLEOTIDASE

Alpha napthly acetate

Adenosine triphosphates

Immuno HISTOCHEMICAL TECHNIQUES

This is an immunologic technique that make use of antigenantibody reactions, whereby the site of antigen binding is demonstrated by an antibody ( direct or indirect)

Polycolonal anitbody

Monoclonal antibody

ANTIGEN RETRIEVAL

Proteolytic Enzyme Digestion

Microwave Antigen Retrieval

Pressure Cooking Antigen Retrieval

E P O S

involves a labeled antibody reacting directly with the antigen in tissue sections new direct technique -large number of primary antibody and peroxidase attached to dextran polymer backbone - reduced incubation time

LSAB (labelled streptavidin biotin and ABC mtd

involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) which reacts with the primary antibody biotin low molecular vitamin that is easily conjugated with enzymes and tumor markers Avidin is found predominately in the egg white of birds, amphibia, and reptiles.

Strepavidin is produced by the bacterium Streptomyces avidinii

PAP

Indirect anitbody-complex where the soluble peroxidaseantiperoxidase complex is bound to unconjugated primary antibody by a second layer of antibody that then binds both the primary and antibody and the PAP complex

Utilize enzyme substrate reaction to convert colorless chromogens into colored end products.
Horseradish peroxidase
-this enz will produce an insoluble dark brown reaction once

combined with DAB ( IF with the presence of an anitgen)

Alkaline phosphatase
-visualized by using either a blue dye (Fast Blue BBN) or a

red dye (Fast Red TR).

-recommended with Blood and bone marrow smears.