This document provides an overview of immunohistochemistry (IHC), including the principle, general protocol, and key steps. IHC combines histology and immunology to identify specific tissue components using antigen-antibody reactions tagged with visible labels. The protocol involves fixing, embedding, sectioning tissues, performing antigen retrieval, incubating with primary/secondary antibodies, and visualizing the antigen-antibody complex through detection systems like peroxidase or fluorescence. Proper controls and interpretation by a pathologist are needed for accurate results.

1. Immunohistochemistry
Presentation
Markos Tadele
Tuesday, April 26, 2016

2. Contents
• Introduction
• Principle
• Method
• General IHC protocol
• Immunostaining

3. 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.

4. 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.

5. ANTIGENS AND ANTIBODIES
Antigens
• 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.

6. 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

7. 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.

9. 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 of
an IgG molecule.

10. 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.

11. Antibody

12. 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.

13. 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

14. 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.

15. 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

16. 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

17. 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

18. 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 produce
Hybridomas 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
antigen
Recognize only one epitope on an antigen
Can have batch-to-batch variability
Once a hybridoma is made, it is a constant
and renewable source

19. 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.

20. 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).

27. 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.
Fixation
The 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 Mounting
Formalin-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.

28. 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.

29. 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.

30. 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

31. 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.

32. 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.

33. 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”)

34. 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

35. 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.

36. 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.

37. 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.

38. 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.

39. 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)

40. detection / Immunostaining
fluorescent technique
Direct method indirect method

41. 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

42. 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.

43. 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

44. 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

45. 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:

46. Controls
• for interpretation and accuracy
– Positive
– Negative