٢٠/٠٧/١٤٣٧

١

ImmunohistochemistryPrinciples and Applications

Prepared by:Dr/ Ahmed Khalaf AbdelhamidLecturer of Poultry diseases, Faculty of Veterinary medicine, AssiutUniversity

Introduction• Immunohistochemistry (IHC) combines histological,

immunological and biochemical techniques for the

identification of specific tissue components by means of

a specific antigen/antibody reaction tagged with a visible

label.

• IHC makes it possible to visualize the distribution and

localization of specific cellular components within a cell

or tissue.

٢٠/٠٧/١٤٣٧

٢

Immunohistochemistry utilizes labeled antibodies

to localize specific cell and tissue antigens, and is

among the most sensitive and specific

histochemical techniques.

HISTORY OFIMMUNOHISTOCHEMISTRY

٢٠/٠٧/١٤٣٧

٣

1945 – Albert Coons 1st used an Ab labeledwith a fluorescent dye to visualize tissues.

1966 – 1st developed enzyme labeling insteadof fluorescent label.

1974 – IHC was performed for the 1st time onroutine formalin fixed paraffin embeddedsections.

1981 – developed avidin-biotin labeling

1991 – Heat induced antigen retrievaltechnique in IHC was done

1995 – Polymer technology introduced

٢٠/٠٧/١٤٣٧

٤

TERMINOLOGIES

ANTIGENS - Molecules that induces formation ofan Ab and is foreign to the animal into which it isintroduced

Sites on Ag that are capable of inducing Abformation are known as – EPITOPES/ ANTIGENICDETERMINANT – the exact site on the Ag withwhich the Ab combines

•ANTIBODIES – IgG is the most frequently used Ab forIHC•The paratope of Ab binds to the epitope of Ag

•Abs are also proteins - thus any part of the Ab mayitself serve as epitope to induce Ab formation (to whichsecondary Ab binds)

•IHC technique prove that Ig molecules can serve bothas Ab and Ag

٢٠/٠٧/١٤٣٧

٥

Formalin fixed paraffinembedded sections

Frozen sectionsSmearsImprints

Cytospins

IHC CAN BEPERFORMED ON -

METHODS

• One step staining method• Labeled Ab reacts directly

with Ag in tissueDIRECT

• Unlabeled primary Abreacts with tissue Ag

• Conjugated second Abreacts against primary Ab

INDIRECT

٢٠/٠٧/١٤٣٧

٦

1. Peroxidase-antiperoxidase method (PAP)2. Biotin-avidin complex method (ABC)3. Labeled streptavidin-biotin method (LSAB)4. Alkaline phosphatase- anti alkaline phosphatase

methos (APAAP)5. Polymer based labeling

Direct Method

It has the advantages of rapidity, ease ofperformance and limited nonspecific reaction.

73

Enzymeor fluorescentprimary Ab

antigen

٢٠/٠٧/١٤٣٧

٧

Indirect Method - Procedure

An unlabeled primary antibody binds tothe tissue antigen.

77

primary Ab (mouse)

antigen

Two-Step Indirect Method

An enzyme-labeled secondary antibodybinds to the primary antibody.

78

secondary Ab(rabbit anti-mouse)

Enzyme orfluorescent

٢٠/٠٧/١٤٣٧

٨

Multiple ImmunolabelingMultiple Immunolabeling

IHC PROTOCOL

Fixation and processing

Section cutting

Deparaffinisation and rehydration

٢٠/٠٧/١٤٣٧

٩

Blocking endogenous peroxidase

Blocking non-specific antibody binding

Antigen retrieval

Primary antibody

Secondary antibody

Chromogen

Chromogen enhancement

Counterstain

Mount

Stringent washingbetween reagents

٢٠/٠٧/١٤٣٧

١٠

Avidin-Biotin Methods• Uses the strong and high affinity of avidin (egg white

glycoprotein) for biotin (water-soluble vitamin).

• Avidin has four binding sites for biotin but fewer thanfour molecules of biotin will actually bind to avidin.

100

avidinbiotin

molecules

Avidin-Biotin Methods

• Two of the most common methods include• Avidin-Biotin enzyme Complex (ABC)• Labeled StreptAvidin-Biotin (LSAB)

101

٢٠/٠٧/١٤٣٧

١١

• The enzyme complex is prepared by mixingbiotinylated enzyme (HRP or AP) and avidin.

ABC Method

avidin-biotin-enzyme complex

103

This preformed avidin-biotin-enzyme complex then reacts with thebiotinylated secondary antibody.

avidinbiotinylatedenzyme

ABC - Procedure

An unlabeled primary antibody bindsto the antigen.

104

antigen primary Ab(mouse)

٢٠/٠٧/١٤٣٧

١٢

ABC - Procedure

A biotinylated secondary antibodybinds to the primary antibody.

105

secondary Ab(rabbit anti-mouse)

biotin

ABC - Procedure

A preformed avidin-biotin-enzymecomplex solution is added and binds tothe biotinylated secondary antibody.

106

avidin-biotin-enzyme complex

٢٠/٠٧/١٤٣٧

١٣

ABC - Procedure

A substrate-chromogen solution isadded ending the reaction andproducing a colored end-product.

107

substrate-chromogen

substrate-chromogen

LSAB Method

• Uses enzyme-conjugated streptavidin.Streptavidin is conjugated to severalmolecules of enzyme horseradish peroxidase(HRP) or alkaline phosphatase (AP).

• The secondary antibody is conjugated tonumerous biotin molecules, each of which canpotentially bind to an enzyme-conjugatedstreptavidin.

109

٢٠/٠٧/١٤٣٧

١٤

LSAB – Procedure

An unlabeled primary antibody bindsto tissue antigen.

110

antigen

primary Ab

LSAB – Procedure

A biotinylated secondaryantibody binds to theprimary antibody.

Each secondary antibodycontains multiple biotinmolecules; severalsecondary antibodies canbind to the primaryantibody.

111

biotin

secondary Ab

٢٠/٠٧/١٤٣٧

١٥

LSAB – Procedure

An enzyme-labeled streptavidin isadded and binds to the secondaryantibody. HRP-streptavidin

112

LSAB – Procedure

A substrate-chromogen solution isadded producing a colored end-product.substrate-

chromogen

113

٢٠/٠٧/١٤٣٧

١٦

LABELS

ENZYMELABELS

COLLOIDALMETAL LABELS

FLUORESCENTLABELS

RADIOLABELS

ENZYME LABELS

•Mc used labels in IHC are enzymes

•Enzymes used are –

HORSERADISH PEROXIDASE (HRP)

ALKALINE PHOSPHATASE (CALF INTESTINAL)

GLUCOSE OXIDASE

β-D GALACTOSIDASE (BACTERIAL DERIVED)

٢٠/٠٧/١٤٣٧

١٧

CHROMOGENS

AEC and DAB

AEC chromogenMart-1

Melanoma

DAB chromogenMart-1

Melanoma

66

Examples of staining results using AEC and DABchromogens.

٢٠/٠٧/١٤٣٧

١٨

Polyclonal Antibodies

22

Polyclonal antibodies reacting with various epitopesEach antibody is made by a different B-cell

Monoclonal Antibodies

Monoclonal antibodies reacting with similar epitopes

32

٢٠/٠٧/١٤٣٧

١٩

APPLICATION OF IHC IN ROUTINESETTINGS

DIAGNOSIS OF TUMORS

PROGNOSTIC MARKER

PREDICTIVE OR THERANOSTIC MARKERS

IDENTIFICATION OF INFECTIOUS ORGANISMS

DIAGNOSIS OFTUMORS

1. Maximum utility of IHC is in distinguishingcarcinoma from lymphoma, sarcoma and melanoma

2. Workup of hematolymphoid neoplasms3. Metastatic carcinoma of unknown primary (CUP)4. Soft tissue neoplasms – 4 common diagnostic setting

a. Small round cell tumorsb. Monomorphic spindle cell tumorsc. Epithelioid soft tissue tumorsd. Pleomorphic spindle cell tumors

٢٠/٠٧/١٤٣٧

٢٠

5. In bone – to differentiate primary from metastaticnon –osseous tumors

6. CNS tumors7. Germ cell tumors

1. Loss of myoepithelial or basal cells orbasement membrane/collagen type IV – theseallow assessment of microinvasion

2. Endothelial markers – assist in identification oflymphovascular spaces to ascertain tumorembolism

3. ER, PR and her2/neu4. Ki-67 /MIB-1 – proliferation markers

PROGNOSTICMARKERS

٢٠/٠٧/١٤٣٧

٢١

PREDICTIVE ORTHERANOSTICMARKERS

1. ER/PR – tamoxifen in Ca. breast2. Her 2 – herceptin in breast cancer3. C-kit – gleevac/imatinib in GIST, CML4. CD20 – rituximab in B-cell NHL5. EGFR – erlotinib in lung cancer

IDENTIFICATION OFINFECTIOUSORGANISMS

1. Viruses – HSV, CMV, EBV2. Others – toxoplasma, pneumocystis