Immunostain and Histochemistry

8/12/2019 Immunostain and Histochemistry

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IMMUNOSTAIN AND HISTOCHEMISTRY

DEFINITIONS

Immunostainingused to localize

specific molecules in tissues. It is based

on antibody-antigen recognition.

Antibody (Ab)also known as

immunoglobulin. IgG is the most

commonly used antibody in

immunostaining.

Antigen (Ag)a substance which

evokes the production of one or more

antibody.

Epitopestructural part of the antigen

that reacts with the antibody

HISTORY

1941Albert Coons first introduced the

use of immunofluorescence as initial

attempts to label antibodies but wasnt

successful because the labels werent

visible under the microscope.

1967Nakane and Pierce developed

the Enzyme-labelled antibody

technique by labelling an antibody with

an enzyme.

1970Sternberger discovered theperoxidase-antiperoxidase (PAP)

method.

1990s Antigen retrieval; it increased

the detection of antigens and sensitivity

of methods.

It was known that the retrieval of

nonreactive antigens in formalin-fixed,

paraffin embedded tissues was possible

through heating sections in buffer

solutions.

IMMUNOHISTOCHEMISTRY (IHC)

combination of immunologic and

histochemical techniques which allow

phenotypic markers to be detected and

demonstrated under the microscope

antibodies are cross-linked to enzymes

which can produce a colored end

product

uses polyclonal, monoclonal,

fluorescent labeled or enzyme-labeled

antibodies

immuno = antibodies ; histo = tissue

Antibody Characteristics

1. Monoclonal - identical copies of the same

unique antibodies, recognizes only a single

epitope.

- highly specific

- intrinsic cross-reactivity to

non-target can be problematic.

- potential for epitope loss

resulting in loss of staining.

- unlimited supply.2. Polyclonal - a mixture of multiple antibodies

recognizing different epitopes of the antigen.

- in general, does produce stronger

signal greater potential for false positive

staining due to Ab cross reacting to others.

- limited supply.

Tissue Preparation for IHC

tissue must be:

a. prepared in a cryostat section

b. fixed in absolute methanol or acetonefor a few second

purpose: prevent destruction of some

of the labile antigenic sites

Sample Preparation:

1. Fixation

To prevent bacterial decomposition

To prevent autolysis by rapidly

terminating the enzymatic/metabolic

activities Used to immobilize antigens while

retaining cellular and subcellular

structures through cross-linking

methylene bridges and Schiff bases

between basic amino acid residues of

proteins.


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Tissue fixation is optimized for

maintaining morphology/ histochemical

staining

Characteristics of Fixatives:

- Acts Rapidly

- Safe/easy to use

- Inexpensive

- Preserves cellular and tissue structures

without introducing artifacts

- Stabilizes tissue, allows for handling and

processing

*The ideal fixative does not exist.

Fixatives:

- Aldehydes (formaldehyde,

glutaraldeyde)- Oxidizing agents, metallic ions and

complexes (osmium tetroxide, Chromic

acid, Zn)

- Protein denaturing agents (acetic acid,

methanol, ethanol)

- Poorly-Understood mechanisms

(mercuric chloride, picric acid)

- Other: microwave,vapor fixation

- Combinations of above

2. Embedding(Frozen Section)

for quick processing and situations

where the antibody may not be

compatible with paraffin compounds

are typically used for direct or indirect

immunofluorescence detection

methods

thickness: 10 microns

(Paraffin)

is used for maximum tissuepreservation and makes a stronger

block for sectioning tissue

thickness: 3-5 microns

3. Retrieval of Masked Antigens

Unmasking the antigen is sometimes

necessary because paraffinization and strong

fixations may inhibit the primary antibodies

attachment to the antigen.

Proteolytic Enzyme Digestion

used in formalin fixed paraffin

sections

trypsin andprotease are the

enzymes commonly used

useful for demonstrating heavy

chain immunoglobulins,

complement and specific

antigens (i.e. cytokeratin)

Microwave Antigen Retrieval

involves boiling of formalin-

fixed deparaffinized sections in

solutions such as:

a. 0.01 M-citrate buffer (pH 6.0)b. EDTA (pH 8.0)

c. Tris EDTA (pH 9.9 or 10.0)

most satisfactory exposure time

on heat: 20 minutes

Pressure Cooking Retrieval Antigen

less time consuming

allows more consistent

recovery of many antigens

4. Immunostaining

used to localize specific molecules in

tissues. It is based on antibody-antigen

recognition.

Detection is commonly achieved using a

colorimetric (enzyme) reaction or a

fluorescent signal generated from an

ultraviolet source.

Purposes:

It determines when and where the

biomarker is expressed.

It helps confirm the genotype and

phenotype.

For co-expression or co-localization.

Cell type-specific biomarkers.


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METHODS IN IHC

1. IHC Controls

used to test the specificity of the

antibodies involved and to avoid

misinterpretations due to false (+) or

false (-)

Positive Control

- a section that is known and

proven to contain the antigen in

question

Negative Control

-omits the primary antibody

from the staining schedule or

replacing the specific primary

antibody by an immunoglobulin

that is directed against anunrelated antigen

Internal Tissue Control

- also called as "built-in control"

-eliminate the variable of tissue

fixation between specimens

and controls

-contains the target antigen

2, Enzyme Labeling

incubated with chromogen to produce astable colored reaction

optimal incubation time: 30-60 minutes

at room temperature

enzyme labeling of Ag with

horseradish peroxidase

staining with chromogen

mixture (i.e. diaminobenzidine

DAB)

insoluble dark brown end

product

3, Direct Technique of IHC

conjugates the primary antibody

directly to the label

advantage:

simple and quick

requires only one application of

the reagent

disadvantage:

less sensitive

has risk of not detecting small

amounts of Ag

4. Indirect Technique of IHC

two or three step procedure that

involves application of the

unconjugated primary antibody and a

labeled antibody directed against the

first antibody

advantage:

inexpensive

more sensitive Horseradish peroxidase

-most commonly used enzyme for

indirect antibody complex techniques

5. Peroxidase-Antiperoxidase (PAP) Technique

exposes an unlabelled primary Ab to

the Ag in the sample and is followed by

removal of any excess primary

antibodies thus allowing secondary

antibody to be introduced in the sample

6. Avidin-Biotin Complex (ABC) Techniques


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uses avidin

- derived from egg white

- high affinity for biotin

-low molecular

weight vitaminthat can be easily

conjugated to Ab

and enzyme markers

staining sequence:

primary antibody

biotylynated secondary

antibody

(strept) avidin-biotin-enzyme

complex or enzyme labeled

streptavidin

7. Labeled Streptavidin Avidin Biotin Technique

(LSAB) Procedure)

4-8 times more sensitive than the old

ABC method

replaced avidin to streptavidin

Staining sequence:

rabbit or mouse antibody

biotinylated anti-rabbit or anti-

mouse immunoglobulin

strepatvidin enzyme conjugate

References:

Gregorios, J. (2006) Histopathologic

Techniques 2nd ed.

Meeker, K. (2012) Immunostaining 101,

http://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/2012/18_Me

eker_IHC_101_9pH.pdf

Boenisch T, et. al,(2009)

Immunohistochemical Staining

methods, 5th ed
http://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/2012/18_Meeker_IHC_101_9pH.pdfhttp://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/2012/18_Meeker_IHC_101_9pH.pdfhttp://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/2012/18_Meeker_IHC_101_9pH.pdfhttp://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/2012/18_Meeker_IHC_101_9pH.pdfhttp://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/2012/18_Meeker_IHC_101_9pH.pdfhttp://www.hopkinsmedicine.org/mcp/PHENOCORE/CoursePDFs/2012/18_Meeker_IHC_101_9pH.pdf

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Immunostain and Histochemistry