TECHNICAL ASPECTS OF

IMMUNOHISTOCHEMISTRY

ANJAN KUMAR.K.R.
PhD Scholar
Dept. of Veterinary Pathology
Veterinary College, Bangalore
KVAFSU

What is immunohistochemistry

Refers to the process of localizing proteins in cells of a tissue section

exploiting the principle of antibodies binding specifically to antigens in
biological tissues.

Immunohistochemistry is a molecular technique that combines principles

from both immunology and biochemistry techniques and principles to the
study of histology and pathology by revealing molecules and patterns within
cells and tissues.

Histochemical localization of immunoreactive substances using labeled

antibodies as reagents.

History:

1942 - direct immunofluorescence

1959 dense-molecular conjugate [Ferritin]
1965 - opaque heavy metal technique [Uranium]
1966 - enzyme tagging method [HRP ]
1967 - immunoperoxidase
1970 - (PAP) technique
1971 - opaque heavy metal technique [colloidal gold]
1974 - avidin- anti - biotin complex (ABC) technique

Applications of immunohistochemistry
Histogenesis and differential diagnosis of
neoplasms
Likely site of origin in adenocarcinomas
Quality assurance in sentinel lymph node
biopsies
Definition of transformation pathways
Detection of microorganisms
Prognostic and predictive markers

Principle of Immunohistochemistry:

antigen

IMMUNOHSITOCHEMISTRY STEPS
Fixation
Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Blocking background staining
Primary antibody
Secondary antibody
Chromogen Substrate
Counterstain
Mounting

Microscopy
Slide 3 Observation
of 23

Fixatives
For preservation of cells and tissues in as
reproducible and life like manner as possible.
They denature proteins by coagulation or by
forming additive compounds and may be
both.
.

Cross linking or non coagulating

fixatives

Formaldehyde
Paraformaldehyde
Glutaraldehyde
NBF

FORMALIN SUBSTITUTES
Bouins solution: deleterious effect on
CD5,CD10&cyclin
B5 lymph node biopsies, recommended for
cytoplasmic antigen detection,CD5 IS
AFFECTED
Zenkers solution- poor penetration,
Hollandes fixative- not a good fixative for
immunohistochemistry
Zinc formalin-for nuclear morphology, tissues
do not adhere to slides

Coagulative fixatives
Ethanol
Acetone

Tissue adhesives

Poly- l lysine coating

Silanized slides - aminopropyltriethoxysilane
Gelatinized slides
VECTABOND

Deparaffinization
Xylene + alcohol
Xylene substitutes
One step deparaffinization and antigen retrieval
method

Antigen retrieval
Is a high temperature heating method to
recover the antigenicity of tissue sections that
had been masked by formalin fixation
AR is influenced by temperature and Ph.

Principle
Calcium induced modification of protein
conformation
Calcium formed a complex with proteins in
formalin fixed tissues leading to antigen
masking
So high temperature and calcium chelation is
required to release calcium form this cage like
calcium complex
Ultimately involves re naturation or at least
partial restoration of proteins to its native state
by hydrolysis of cross links driven by heat

AR METHODS
PIER- protease induced epitope retrieval
Other enzymes like: trypsin, proteinase,
pronase, pepsin
Acts by digestion of proteins but cleavage is
non specific and some antigens may be
negatively affected.
Has low no. of antigens for which this method is
optimal

AR
HIER involves placing slide in heated solutions of
various composition before application of antibody
Involves shorter heating times with high temperatures
Methods: microwave, vegetable steamer, pressure
cooker

TYPES OF AR SOLUTIONS
0.1M Citrate buffer solution pH: 6.0
O.1M EDTA buffer solution pH: 8.0
0.5M Tris base buffer pH: 10.0

Effect of HIER on tissues other than AR

Enhance endogenous biotin in tissues.
use of citrate buffer reduces endogenous biotin
activity

Blocking of endogenous enzymes

Peroxidase
Alkaline phosphatase
3% H2O2 in deionized water or methanol
applied on sections inactivates endogenous
peroxidase.
ALKALINE PHOSPHATASE- can be blocked
using LEVAMISOLE

Blocking with normal serum

Usually the serum is taken from the same
species used for production of secondary
antibodies.
Usually done before incubation of primary
antibody.
Done to prevent non specific binding of
secondary antibody.

Blocking of endogenous biotin

Can be blocked by using AVIDIN in PBS for 20
min ( 0.05 0.2 %) when using ABC method
Done whenever ABC Method is used.
Can be avoided by using avidin biotin free
detection system like secondary polymer based
antibodies

Washing buffers
Most laboratories employ PBS / TBS.
Adding tween 20 prooves to be more effective.
However PBS is not recommended when using
Alkaline phosphatase based detection system.

ANTIBODIES
Most commonly used are IgG
IgM is less commonly used.

TYPES OF ANTIBODIES
Monoclonal
polyclonal

POLYCLONAL

MONOCLONAL

Antigen
An antigen is a foreign substance that stimulates antibody
formation and has the ability to bind to an antibody.

The site on an antigen to which a

complementary antibody may specifically react
is called an epitope or antigenic determinant.
antibody

Antigen Antibody reaction

Antigen antibody reaction

Antibodies bind to antigen through the variable
regions of the antibody. This bonding may be
hydrophobic, ionic, Van der Waals or hydrogen
bonding.
The strength of the binding of an antibody to a
specific antigen is called affinity.
High affinity antibodies will bind larger amounts
of antigen in a given period of time, and can be
used at higher dilutions.

Handling of antibodies
Storage and containers used:
Should have negligent protein adsorptivity
Poly propylene or borosilicate glasses.
Storage temperature:
Ready to use antibodies are stored at 2-8 C
Cocentrated antibodies are aliquoted and
stored at -20C

Titration of antibodies for

immunohistochemistry
Titer refers to extent to which a particular
solution is diluted by adding another solution.
If we are starting with 1 ml of antibody and want
to dilute all of it by 1:10, we would place the 1
ml of antibody into a test tube (that holds at
least 10 ml), and add 9 ml of diluent, so that our
new final volume is 10 ml.

 For example, to make 1 ml (final volume) of a 1:100

titer of a primary antibody, we would take 0.01 ml of
primary antibody, and add diluent to a total final
volume of 1 ml (so we would be adding 0.99 ml of
diluent).
usually try to have an initial final volume of 0.5 ml or 1
ml. For example, if we have to prepare a titer of an
antibody at 1:100, we can do either of the following:
For a final volume of 0.5 ml:Take 5 ul of primary
antibody, add 0.495 ml of diluent. For a final volume of
1.0 ml:Take 10 of primary antibody, add 0.99 ml of
diluent

Preparation of serial dilution

start with an initial volume of 1 ml of solution
prepare 1 ml of a 1:100 dilution
sake of discussion lets name it as test tube A
Next, take 0.5 ml of this solution, and place it in another
test tube (named B), and then add 0.5 ml of diluent to
test tube B and mix. Test tube B now has a 1:200
dilution of the primary antibody.
Next, take 0.5 ml from test tube B and add it to another
test tube (C). Add 0.5 ml of diluent to test tube C, and
we now have 1 ml of 1:400 primary antibody solution in
test tube C.

1:50

1:400

1:100

1:800

1:200

Incubation of primary antibodies

Depending on the
antibody it could be
30min to over night.
However optimum results
are achieved at 25 C
Humidity chambers must
be used when incubating
at higher temperature to
prevent drying of tissue
sections

SECONDARY ANTIBODIES
Monospecific antibodies recognize antibodies
raised in one species
Multi link secondary antibodies- universally
recognize both mouse and rabbit antibodies
and are based on polymer back bone.
Conjugated secondary antibodies.

(2)
Polymer based secondary antibody

(1)

antibody 1, mouse

METHODS OF
IMMUNOHISTOCHEMISTRY

Direct method

enzyme

primary Ab

antigen

An enzyme-labeled primary antibody

binds to the tissue antigen.

A substrate-chromogen solution is added

producing a colored end-product.
substrate-chromogen

Indirect method:
Two step indirect method
Three step indirect method

Two-Step Indirect Method

Uses an enzyme-labeled secondary antibody that is
directed against the unlabeled primary antibody.
If the primary antibody (which is now the antigen) is
made in mouse, the secondary antibody must be against
mouse immunoglobulin.
More sensitive than the Direct Method because several
secondary antibodies are likely to bind with a number of
various epitopes on the primary antibody increasing the
enzyme labels involved.

An unlabeled primary antibody binds to the tissue

antigen.

primary Ab (mouse)

antigen

An enzyme-labeled secondary antibody

binds to the primary antibody.
secondary Ab
(rabbit anti-mouse)

enzyme

A substrate-chromogen solution is added

producing a colored end-product.

substrate-chromogen

 Only one secondary antibody is shown bound to the

primary antibody in the previous illustration. However,
several secondary antibodies are likely to bind with
various epitopes on the primary antibody, thus increasing
the signal.

Three-Step Indirect Method

Uses another layer of enzyme-labeled (tertiary)
antibody that is added to the previously
described Two-Step Indirect Method.
The added antibody layer increases signal and
staining intensity that is helpful when staining
antigens with few or limited available epitopes.
Both secondary and tertiary antibodies must be
conjugated to the same enzyme.

An unlabeled primary antibody binds to

the tissue antigen.

primary Ab (mouse)

antigen

Three-Step Indirect Method Procedure

An enzyme-labeled secondary antibody binds to the primary antibody.

Soluble enzyme immune complex techniques

PAP method
APAAP method

 These methods are among the sensitive IHC

techniques, attributed to more enzyme molecules
being localized per antigenic site.
Methods include
peroxidase-antiperoxidase (PAP)
alkaline phosphatase-antialkaline phosphatase
(APAAP)
The secondary antibody must be directed against both
the primary and the antibody of the enzymeantienzyme immune complex.

Preformed soluble enzyme-anti enzyme immune complex is prepared by mixing

excess enzyme to its antibody.

Peroxidase anti-peroxidase method PAP immune complex consists of

three molecules of peroxidase and two antibodies

APAAP Method APAAP immune complex consists of two molecules of

alkaline phosphatase and one antibody.

(Strep) avidin biotin method

Uses the strong and high affinity of avidin
(egg white glycoprotein) for biotin (watersoluble vitamin).
Avidin has four binding sites for biotin but
fewer than four molecules of biotin will
actually bind to avidin.

 Two of the most common methods include

Avidin-Biotin enzyme Complex (ABC)
Labeled StreptAvidin-Biotin (LSAB)

The inherent amplification of sensitivity offered

by avidin and biotin makes these methods
more favorable than PAP or APAAP.
Streptavidin, a bacterial protein has recently
replaced avidin because it produces less
background staining than avidin.

Avidin has four binding sites for biotin

but fewer than four molecules of biotin
will actually bind to avidin.

 The enzyme complex is prepared by mixing

biotinylated enzyme (HRP or AP) and avidin. This
preformed avidin-biotin-enzyme complex then reacts
with the biotinylated secondary antibody.

ABC - Procedure

ABC Method with CSA

LSAB METHOD
Uses enzyme-conjugated streptavidin.
Streptavidin is conjugated to several molecules
of enzyme horseradish peroxidase (HRP) or
alkaline phosphatase (AP).
The secondary antibody is conjugated to
numerous biotin molecules, each of which can
potentially bind to an enzyme-conjugated
streptavidin.

biotinylated secondary antibody binds to the primary antibody.

Each secondary antibody contains multiple biotin molecules;
several secondary antibodies can bind to the primary antibody.
Enzyme-labeled streptavidin is added and binds to the secondary
antibody

Double immunohistochemistry

Enzyme - chromogen combinations

Spectral imaging

Spectral imaging

QUALITY CONTROL: Use of tissue

microarray control blocks:

Multi tumor sandwich blocks (MSB's) have many uses in quality

control.
Their use in assessment of optimal titers of primary antibodies.
They also provide a rapid and efficient way to screen unusual or
new antibodies for sensitivity and specificity, and to compare them
with current similar reagents.
They provide an ideal preparation for use as positive control

sections.

The Use of Vimentin in Quality

Control

there are always some vimentin-containing cells

in nearly any tissue taken for biopsy purposes
If the vimentin stain shows very weak staining
on a case or in a particular area on the slide, it is
probable that these areas have been over fixed
in formalin and are showing the effects of
antigenic damage.
As such, one needs to keep in mind the degree
of antigen damage when assessing the results
of the other stains.

Control:
Negative control:
using only one type of epitope retrieval for all
slides, only one negative control slide
needs to be run for every different tissue
block that is stained.
It is particularly important that the
negative control slide be subjected to
the same epitope retrieval procedures as
the primary antibody, since these epitope
retrieval procedures can markedly intensify
endogenous biotin artifacts.

Positive Control Slides:

A positive control section needs to be run with every
different antibody that is used. The positive control
section should be mounted on the same slide as the
tissue section being stained, as that is the only way to
assure that the case tissue and the control tissue are
handled in the same fashion.
multitissue sandwich block preparations should be
used as positive control sections.
. These preparations allow you to assess multiple
known positive tissues and also multiple known
negative tissues

ARTIFACTS IN IHC

Desquamartifact
"Bubble" artifacts
Drying artifacts
Trapping artifacts
Edge artifacts
Artifacts of poor fixation.
Bacterial contamination artifacts
Endogenous biotin artifacts
Graphite pencil artifacts

CONCLUSION:
logical differential diagnosis based on the
clinical and morphologic findings.
garbage in, garbage out.
IHC is not perfect and all tumors will not
necessarily react as they are "supposed" to
NEVER use IHC to tell you whether
something is benign or malignant

 Multitumor Block Preparations

There is no such thing as a "Prediluted
Ready-to-Use" antibody.
HIER techniques or proteolytic digestion
Experiment with new methods, fixatives,
reagents,techniques and modifications.
That is the only way that any laboratory will
grow and improve with age.
Use of technology has reduced the time
involved in immunohistochemistry

 Thank you