Immunohistochemistry

Presentation

Markos Tadele

Tuesday, April 26, 2016

Contents

• Introduction

• Principle

• Method

• General IHC protocol

• Immunostaining

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 with

in a cell or tissue.

Principle

• The principle of immunohistochemistry is the

localization of antigens in tissue sections by the

use of labeled antibodies as specific reagents

• antigen-antibody interactions that are visualized

by a marker such as fluorescent dye, enzyme,

radioactive element or colloidal gold.

ANTIGENS AND ANTIBODIESAntigens

• An antigen is a substance foreign to the host which

stimulates formation of a specific antibody and which

will react with the antibody produced.

• Antigens have two main properties.

• immunogenicity, which is the ability to induce

antibody formation.

• specific reactivity, which means that the antigen can

react “with the antibody it caused to be produced.

Antigen

• The reaction between an antigen and its

antibody is one of the most specific in biology.

• Antigen-mainly proteins, glycoprotein,

polysaccharides

• Complementary Determining Region

Antibodies

• An antibody is a serum protein that is formed in response to exposure to an antigen.

• Reacts specifically with that antigen to form immune complexes either in the body or in the laboratory.

• Antibody production is a response by the body to foreign material (an antigen), and is designed to rid the body of this invader.

Antibody Structure• Structurally, an antibody is made up of two kinds of

protein chains - heavy and light chains.

• Immunoglobulins are named for their heavy chains

– IgG molecule have heavy chains of the gamma type.– An IgA antibody has alpha heavy– chains; IgD, delta heavy chains.– IgE, epsilon heavy chains– IgM has mu heavy chains.

• A primary antibody for immunoperoxidase staining that is"specific for gamma chains"will localize the heavy chain ofan IgG molecule.

Light chains

• There are only two types of light chains common to all

five groups: kappa and lambda.

• An IgG molecule has two identical light chains. either

two kappa chains or two lambda chains

• A single antibody can never have both kappa and

lambda chains.

Antibody

Antibody production • A source for the antigen such as serum, urine

or tissue is subjected to a combination of

procedures including precipitation,

centrifugation, dialysis…

• Other techniques like chromatography and

electrophoresis to obtain a highly purified

antigen.

• The antigen is then injected into an animal, not

same species.

Antibody production

• Antibody production begins within twenty

minutes after injection, although a

measurable quantity of antibody cannot be

detected for 5-10 days.

• Sometimes boosting is important

Monocloanal and polyclonal antibodies

Polyclonalantibodies

– Large complex antigens may have multiple epitopes

and elicit several antibody types. Mixtures of different

antibodies to a single antigen are called polyclonal

antibodies.

Monoclonalantibodies

– Antibodies specific for a single epitope and produced

by a single clone are called monoclonal antibodies and

are commonly raised in mice.

Antibody caharacteristics

Polyclonal

1. in general produce stronger signals

2. greater potential for false positive staining

due to antibodies cross-reacting to undesired

targets(affinity purification helps)

3. limited supply

Monoclonal

1. highly specific

2. less background

3. intrinsic cross-reactivity to non-target can be

problematic

4. potential for epitope loss = loss of staining

5. unlimited supply

Polyclonal antibodies are made

by injecting an animal with

your target antigen .

Immune response

Monoclonal antibodies are made

by injecting an animal with

target antigen.

spleen cells Myeloma cells

HGPRT-

fused

Monocloanal and polyclonal antibodies

Summary

Polyclonal antibodies Monoclonal antibodies

Inexpensive to produce Expensive to produce

Skills required for production are low Training is required for the technology used

Relatively quick to produceHybridomas take a relatively long time to

produce

Generate large amounts of non-specific

antibodies

Generate large amaounts of specific

antibodies

Recognize multiple epitopes on any one

antigenRecognize only one epitope on an antigen

Can have batch-to-batch variabilityOnce a hybridoma is made, it is a constant

and renewable source

Conjugation/ labelling

• Antibodies are not visible with standard

microscopy and must be labeled in amanner

that does not interfere with their binding

specificity.

• Conjugation is the process of chemically linking some

type of marker onto an antibody molecule.

• A wid e valriety of conjugates are available for use In

various direct and indirect immunohistological stains.

Conjugation/ labelling

• Common labels include:

Fluorochromes (fluorescein, rhodamine)

Enzymes demonstrable via enzyme

histochemical techniques (peroxidase, alkaline

phosphatase)

electron scattering compounds for use in

electron microscopy (ferritin, colloidal gold).

Step Effect on IHC

Biopsy Depending on the appropriate tissue type, tissue

samples can be obtained in different ways such as

punch/core biopsy, excisional/incisional etc.

Tissue degradation begins at the time of sample

removal.

FixationThe sample should be fixed as soon as possible after

sampling, ideally within less than an hour. The chemical

fixation crosslink proteins in the sample thereby

stopping the degradation process.

Embedding After fixation, the sample is embedded in paraffin for

long-term storage and to enable sectioning for

subsequent staining. Once embedded in paraffin,

samples can be stored (almost) indefinitely.

Sectioning

and MountingFormalin-fixed, paraffin-embedded tissues are sectioned

into thin slices (4-5 μm) with a microtome.

The sections are then mounted onto adhesive-coated

glass slides.

Antigen

Retrieval Due to the fixation process, an antigen retrieval

treatment is applied to unmask the epitopes, either by

heat (heat-induced epitope retrieval; HIER) or

enzymatic degradation (proteolytic-induced epitope

retrieval; PIER).

Primary

Antibody The specificity and sensitivity of the antibody affect the

staining result.

Visualization The antigen/antibody complex signal is amplified and

visualized using a detection system. The strength of

amplification of the reaction affects the staining result

(intensity).

Interpretation

The staining pattern is assessed by a pathologist in

context with other biomarkers, controls and other tests

(e.g. H&E, special stains.

Fixation

Goals of fixation

• Prevent autolysis by rapidly terminating

enzymatic/metabolic activities

• Prevent bacterial decomposition.

• Preserve tissue structures while stabilizing and

hardening the tissue for processing.

Slide Preparation

IHC-P: Paraffin-embedded Cell Pellets and Tissue

• Prior to immunostaining, harvested and fixed in 10%

neutral buffered formalin (NBF) to preserve cell

morphology and target epitopes.

IHC-F: Frozen Tissue

• Frozen tissue should be stored at -80°C

• When ready to stain, equilibrate tissue at -20°C for 15

minutes before attempting to section. Section the tissue

to a 6-8 μm thickness using a microtome

Slide Storage

• freshly cut slides- For best results

• Slides may lose antigenic potential over time

in storage.

• If slides must be stored, do so unbaked at 4°C.

Deparaffinization/ rehydration

• Paraffin wax must be completely removed- for

staining

• This is done through a series of sequential

xylene/ethanol/water washes that remove the wax

and rehydrate the tissue for subsequent antibody

binding.

• Insufficient paraffin removal can lead to spotty,

uneven background staining.

Retrieving antigens “lost” during fixation

• Frozen sections were commonly used to bypass the

problem of ”over fixation” but gives poor

morphology

• Limited protease treatment could allow successful

antibody staining in previously negative tissues

• Cross links can block antibody access to target

epitopes.

• Treatment with protease can re-expose epitopes

(“antigen retrieval”)

Heat induced antigen retrival

• Heat treatment could dramatically improve

immunostaining results: HIER (heat induced

epitope retrival)

• Used with different buffers

• microwave

• heat plate (immersion)

• steamer

• pressure cooker/autoclave

Enzymatic Antigen Retrieval

• Antigen retrieval can also be achieved through enzymatic digestion with pepsin, trypsin, or proteinase K.

• For those antibodies that require enzymatic retrieval rather than HIER, the recommended enzyme and digesting conditions will be clearly indicated on the product datasheet.

Primary antibody

Antibody Incubation

• overnight primary antibody incubation at 4°C,

and all is recommended

Washing

• Adequate washing is critical for contrasting low

background and high signal.

• Wash slides three times for 5 minutes with water

or other solvent after antibody incubation.

Blocking

• For IHC-P- recommended

• blocking the samples in buffered saline

plus 5% normal goat serum (NGS) for 1

hour at room Temperature

• prevent nonspecific background

staining. Block samples.

Counterstains

• After antibody detection but before cover slip it

is advisable to counter stain the tissue to

visualize cellular anatomy and orient the

viewer in respect to the specific staining.

Detection and immunostaining

Avidin biotin-complex (ABC)

• require a 2-step process of detection

involving biotinylated secondary antibody

followed by exposure to an avidin-

peroxidase complex prior to chromogenic

detection.

• liver and kidney that possess high levels of

endogenous biotin, (polymer-based detection

systems)

detection / Immunostaining

fluorescent technique

Direct method indirect method

Immunoflourescent techniques

• Advantages:

– Hi-resolution, easy to double/triple label

– Better sub cellular detail

– Can be used with 3D microscopy/live imaging

• Disadvantages:

– Background Auto fluorescence

– Cost

– Lack of surrounding tissue/cellular detail

– Not permanent

Peroxidase (HRP horse reddish peroxidase)

• producing good sensitivity

• Most commonly used

• Most often used substrate:

• 3,3'-Diaminobenzidine Tetrahydrochloride (DAB)

• DAB reacts with HRP to form a brown precipitate at the

site of antibody binding.

Other Peroxidase Substrates

4-chloro-1-naphthol = BLUE

4-napthol pyronin = RED-PURPLE

2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic

acid) (ABTS)

3-amino-9-ethylcarbazole (AEC) = RED

VECTOR Nova = RED

VECTOR VIP = PURPLE

Enzyme linked immunostaining

• Antibodies cross-linked to enzymes that generate an

intensely colored

Alkaline phosphatase 5-bromo-4-chloro-3-indolyl

phosphate/nitroblue tetrazolium (NBT)

– Vector Red

– Vector Blue

– Vector Black etc

• Glucose oxidase

– with nitroblue tetrazolium

• B-D-Galactosidase

Mounting and examination

• Aqueous and non aqueous (permanent) mounting media are

available.

• The mounting media depends on the chromogen used

during the detection step and its solubility in organic

solvents or water

• Water insoluble Chromogens should not be used with

nonaqueous mounting media andwater soluble chromogens

should not be used with aqueous media

• Nonaqueous mounting media is not compatible with water;

therefore, the samples must be first dehydrated with a

series of ethanol and xylene washes:

Controls

• for interpretation and accuracy

– Positive

– Negative