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HISTOCHEMICAL

and

IMMUNO-HISTOCHEMICAL

TECHNIQUES

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Histochemical stainingtechnique

Tissue constituent Principle Reagents use Resultingcolor

carbohydratesPAS

To oxidize thepolysaccharides andmucin to liberate thealdehyde through the

use of schiffs regent

Schiffs reagent

Nuclei blueblack/blue

Positive subs.red/magenta

proteins Alkalinefast

green

To stain ionized basicgroup such as

protamines andhistones

Fast green

Trichloroaceticacid

green

Cystine andcysteine

Peraceticacid-alcianblue

To oxides cystine andcysteine to form

cysteic acid whichstained blue-green by

a basic dye

Peracetic acid

Alcian blue

Blue-green

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Alkaline fast greenPas positive

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Tissue constituent Principle Reagents use Resultingcolor

Nucleic acid

DNA

Feulgentechnique

to liberate aldehydegroups from the

containing sugar ofthe nucleic acid. It

will then stained theliberated aldehyde

group which isacted upon by theschiffs reagent.

Schiffs reagent DNA REDPURPLE

Cytoplasm -GREEN

RNA ANDDNA

Methylgreen-

pyroninmtd

Anionic phosphategroup of the DNA ishighly selective and

is able to bind to thestained giving theDNA a green color,

while Pyronin will beacted to RNA giving

it a pinkish color.

Methy green

pyronin

DNA(chromatin)

GREEN or

BLUE GREEN

RNA (nucleoli) ROSE RED;

GRANULES(DARK RED

ROSE)

Plasma cell

cytoplasm -PURPLE

Histochemical stainingtechnique

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Feulgen DNA Methyl green pyronin

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Enyzme histochemistry

enzyme mtd principle Reagent use Resultingcolor

Acidphosphatase

Gomori lead Phosphates reacts

on Ca ions to formCa Phosphatewhich is thentreated with cobaltnitrate it willproduce Cophoshates which

gives a blackprecipitate

Sodium -

glycerophosphate

Acid

Phosphatase activityblack

Nuclei -green

5-nucleotidase

Lead Adenosine-5-phosphate ishydrolyzed tophosphate ions inthe presence of Mg.

it is thenprecipitated by leadions to producelead phosphate

AMMONIUM SULFIDE

SUBSTRATE

BROWN

AdenosinetriPHOSPH

ATASE

Metalprecipitation

Uses a substrate toform phosphateions which will thenprecipitated by leadto form leadphosphates

Lead

Ammonium sulfate

Brownishprecipitate

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enzyme mtd principle Reagent use Resulting

color

non-specific

esterase

Indoxylacetate mtd

Uses a substratethat will be thenproduce bromoindoxyl that will bethen oxidized to aninsoluble dye

bromo-indoxyl acetate(substrate)

Esteraseblue

Nuclei - red

Alphanapththy

acetate

Uses substrate thatreleases naptholduring enzhydrolysis and willform an insolubledye at the site of theenzyme activity

a-napththyl acetate(substrate)

Diazonium salt

Esterasereddishbrown

Nuclei -green

monoamineoxidase

Tetrazoliummtd

Demonstrated thruthe oxidation ofTNBT

tetra nitro-bluetetrazolium (TNBT)

Monoamineoxidase

BLUISHBLACK

Enyzme histochemistry

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LEAD METHOD OF 5-NUCLEOTIDASEAlpha napthly acetate

Adenosine triphosphates

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Immuno

HISTOCHEMICAL

TECHNIQUESThis is an immunologic technique that make use of antigen-antibody reactions, whereby the site of antigen binding is

demonstrated by an antibody ( direct or indirect)

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Polycolonal anitbody Monoclonal antibody

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ANTIGEN RETRIEVAL

Proteolytic Enzyme Digestion

Microwave Antigen Retrieval

Pressure Cooking Antigen Retrieval

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EP

OS

involves a labeled antibody reacting directly withthe antigen in tissue sections

new direct technique-large number of primary antibody and peroxidase

attached to dextran polymer backbone- reduced incubation time

LSAB (labelled streptavidinbiotin and ABC mtd

involves an unlabeled primary antibody (first layer)which reacts with tissue antigen, and a labeledsecondary antibody (second layer) which reacts

with the primary antibody

biotin low molecular vitamin that is easily conjugated withenzymes and tumor markers

Avidin is found predominately in the egg white of

birds, amphibia, and reptiles.

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PAP

Strepavidin is produced by the bacterium Streptomyces avidinii

Indirect anitbody-complex where the soluble peroxidase-antiperoxidase complex is bound to unconjugated primaryantibody by a second layer of antibody that then binds boththe primary and antibody and the PAP complex

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Utilize enzyme substrate reaction to convertcolorless chromogens into colored end

products.

Alkaline phosphatase

-visualized by using either a blue dye (Fast Blue BBN) or ared dye (Fast Red TR).

-recommended with Blood and bone marrow smears.

Horseradish peroxidase

-this enz will produce an insoluble dark brown reaction oncecombined with DAB ( IF with the presence of an anitgen)

-