1. Immunohistochemistry
Fundamentals, Pitfalls
and Standardization
NSH October 2007
Lisa Paez, HTL, QIHC (ASCP)
Sherry Smith, HTL (ASCP)

2. IHC fundamentals
1. Basic Immunology, Monoclonal,
Polyclonal and Rabbit Monoclonal
Antibodies
2. Basic IHC techniques,
Pretreatments, Fixation, Processing
and Detection Methods

3. 1.Basic Immunology, Antibodies
and Pretreatments
Attendees will gain a basic knowledge of:
 Antigens and Antibodies
 The concept of the immune reaction
 Tissue fixation and pretreatments

4. What is the Purpose of IHC?
 To identify and localize proteins or
carbohydrates
Human or animal proteins
• In cells or tissues
Bacterial cells
Viral proteins
?

5. Who Cares? Why Localize
Proteins?
 The Central Dogma of Molecular Biology

6. Who Cares? Why Localize
Proteins?

7. How Can We Visualize This
Process?
 Immunohistochemistry- IHC is currently the most
sensitive way to localize proteins within a tissue.
 IHC allows us to stain protein expressing cells, cell
membranes, cytoplasm, nuclei and even organelles.

8. 1.Basic Immunology, Monoclonal,
Polyclonal and Rabbit Monoclonal
Antibodies
 Immunology is the study of the body’s
immune system
 body’s defense system
• responsible for protecting the body from invading
organisms that cause disease
 Many cell types involved in this process
 Belong to the family of blood cells called
white blood cells
 Circulate through the blood and tissues
looking for “foreign invaders”
 When they find something that is foreign,
they will surround it and destroy it.

9. Lymphocytes
 Many types of white blood cells
 White blood cells of interest to
antibody production are the
lymphocytes
All lymphocytes originate in the bone
marrow during fetal development.
 Two main types of lymphocytes
• T-lymphocytes (or T-cells)
• B-lymphocytes (or B-cells)femur

10. Thymus gland
T-Cells
 Lymphocytes that migrate from the
bone marrow to the thymus in order to
mature there
femur

11. B-Cells
only B-cells produce antibodies
 Lymphocytes that remain in the bone
marrow and mature in the bone
marrow

12. + - + + - - - + + - - + - + + - - - + + - -
Proteins
 Proteins are large molecules made up of made
up of smaller molecules called amino acids
 Amino acids have electrostatic properties
(positive, negative and neutral charges) that
determine their interaction with each other.

13. Protein Structure
 The amino acids are first linked together in a simple
chain called the primary structure.
 The amino acids are then further linked into spirals
(helices) or pleats (beta pleated sheets). This called
the secondary structure.
 The secondary structure chains can then be folded
onto themselves and/or linked together to form
tertiary structures.
+ - + + - - - + + - - + - + + - - - + + - -

14.  Large protein molecules (called glycoproteins)
 Belong to the immunoglobulin (immune protein) or Ig
family.
 Produced by B-cells as part of the body’s defense system in
response to a foreign substance.
 Each antibody molecule is made up of 2 different types of
protein chains
 Long chains and short chains
 Long chains are heavier than the short chains
 Referred to as heavy chains and light chains,
respectively
Antibodies

15. Structure of Antibodies
Light chain
(L-chain)
Heavy
chain
(H-chain)
 The protein chains are held together
by disulfide bonds

16. Structure of Antibodies
 Heavy and Light chains are folded
onto themselves in a three-
dimensional structure
gives a definite physical shape to
the antibody molecule
Light chain Light chain
Heavy chain heavy chain
Light chain
Heavy chain

17. Heavy and Light Chains
 There are five types of heavy chains
 They are called by their Greek symbols
∀ γ - gamma
∀ α - alpha
∀ µ - mu
∀ ε - epsilon
∀ δ - delta
 Two types of light chains called by their
Greek symbols
∀ κ - kappa
∀ λ - lambda

18. H-Chains
 Within each class of heavy chains the
proteins are strung along the chain in a
specific sequence.
∀γ heavy chains, α heavy chains, µ heavy
chains, ε heavy chains and δ heavy chains
each have a specific protein sequence.
ε γ α µ δ

19. Antibody Classification
 Each immunoglobulin is composed of at least
 Two heavy (H) chains
 Two light (L) chains
 Antibodies are named according to their heavy chain
composition
 An immunoglobulin with:
 γ heavy chains is called Immunoglobulin G ( IgG )
 α heavy chains is called Immunoglobulin A ( IgA )
 µ heavy chains is called Immunoglobulin M ( IgM )
 ε heavy chains is called Immunoglobulin E ( IgE )
 δ heavy chains is called Immunoglobulin D ( IgD )

20. Antibody molecules
 Antibody molecules vary in size
 Some have multiple sets of heavy and light chains
 IgM is the biggest antibody molecule
 composed of five sets of the two-heavy
chain/two light chain structures

21. Antibody function
 Various classes of antibody molecules have different biological functions
 Effective at detecting different kinds of invading substances
 Involved in various types of immune reactions
-IgM
first antibody type to be produced in the early stages of an immune
reaction
-IgA
produced by lymphocytes that “patrol” the gastro-intestinal tissue
produced in response to a parasitic infection
-IgE
produced by lymphocytes in response to an allergic reaction
-IgG is the most common antibody type circulating in the blood and
tissues.
 Of the five immunoglobulin (Ig) classes, IgG and IgM are the most commonly
used for applications in IHC.

22. Antibody Molecule
 Together, H-chains and L-chains form two functionally different
parts of the the Ig-molecule
 The antigen binding fragment (Fab-portion)
 The crystalline fragment (Fc-portion).
Crystalline Fragment
(Fc )
Antigen binding
Fragments
(Fab )

23. Antibody/Antigen
 Antibodies in circulation and on the surface of B-cells react to foreign
substances (“non-self”)
-Antigen
 To elicit a reaction
-must be of a certain size
-must usually be made up of protein or carbohydrate
 Antibodies do not “recognize” the whole antigen
-React to specific physical and chemical structures on the surface
of the antigen
-Bind to these structures through a “lock-and-key” type fit
-“Closeness” of the fit (or the strength of the binding) depends on
chemical and structural interaction of the antibody and antigen.
 Antigenic site that binds to the antibody is called the epitope or
determinant
 The antibody site or structure that reacts specifically with the epitope is
called the idioptype

24. H-chainL-chain
Antigen
epitope
Antigen Binding Site
 Three-dimensional shape & chemical properties of the
antigen binding region determines what substances will be
able to bind to the antibody.
Antigen binding
Fragment (Fab )

25. Immune Reaction
 The Antibodies can be either:
 Circulating
 Embedded in the cytoplasmic membrane of B-cells
it is the first exposure to antigen ?
If first encountered, there will be no circulating antibodies
Nucleus
Cytoplasm
Cytoplasmic membrane
Antibody molecule
Blood
CirculationLymphatic
Circulation

26. Immune Reaction (First
encounter)
 If antibody has a “good fit” for the antigen
 Will bind to the antibody on the surface of the B-cell
 Trigger the B-cell to produce more of its antibody and release it into
the blood stream
 Released antibodies will then seek out similar antigens and surround
them (opsonization)
 Sends a signal that increases the blood flow and causes other white
cells to migrate to the area
 Other white blood cells (phagocytes) will “chew” up the antigen
(phagocytosis)
 B-cell will reproduce and make many copies of itself (“clones”)
 Clones (activated B-cells) will produce more identical antibodies and
release them into circulation.
 Activated B-cells become super-sensitized to this antigen and have a
very long life-span.("memory” B-cells)

27. Immune Reaction (First
encounter)
1. Antibody on the surface of the
B-cell encounters an antigen
(bacteria cell)
2. B-cells multiply and produce antibodies
3. Antibodies surround antigens
5. Phagocytes migrate to
the area
4. Antibodies bound to the antigen allow the
antigen to be detected by phagocytes
6. Phagocytes engulf and
dispose of antigen
B-Cell
Phagocyte

28. Antibodies
 Generated (directed) against the antigen of
interest, usually a protein i.e.
ER, PR, CD45, HER-2/neu
 Generated by injecting the protein or a
portion of the protein into an animal
 Produced by drawing the blood from the
animal and processing the blood or cells

29. Antibodies
 In order to use antibodies to identify antigens,
the antibody must "recognize" the structure of
the particular antigen
 Characteristics of a "GOOD" antibody:
High Specificity for its target structure
High affinity (stereochemical fit between
antibody and antigen)
High avidity (binding strength between
antibody and antigen)

30. Antibodies
 May be
 Polyclonal
 Monoclonal
 "Cocktail"
 May be from different animal species i.e.
 Polyclonal – rabbit, goat, rat, pig, horse etc.
 Monoclonal – mouse, rat, etc.
 May be whole molecules or fragments
 Available in different formats

31. Polyclonal Antibodies
 Made from serum
 In vivo method of production
 Directed against many epitopes
 Good screening antibodies
 Produced by
• Generating an immune reaction in an animal
• Collecting the blood
• Extracting the serum
 Most commonly used animals for polyclonal antibody
production are rabbit, goat, sheep, horse or donkey
 The immunoglobulin type is most typically IgG
 Many available
 S-100
 Herceptest

32. +Human Antigen Inject into animal
Bleed animal
Extract serum
Containing antibodies
Generate immune reaction with antibody
production
Several Antibodies directed against
several portions of the Antigen
Polyclonal Antibodies
In Vivo Method of Production:

33. Polyclonal Antibodies
 Definition of Serum:
The clear, thin and sticky fluid portion of the blood that remains after
coagulation. Serum contains no blood cells, platelets or fibrinogen
 Extracting Serum:
 Obtain blood from the host
 Allow sample to coagulate
 Centrifuge blood for separation of:
• Cellular components (red & white blood cells)
• Fibrin
• Serum
Red Blood cells
White Blood cells
Fibrin
Serum

34. Polyclonal Antibodies
 Easy to produce, widely available
 Production method results in high yields
 Directed against multiple epitopes, therefore
highly sensitive
Good as "screening" antibodies
 Low specificity, tend to have more background
 Greater variability from lot-to-lot
Consistency relies on availability of the same
animal
 Field is moving towards monoclonals

35. Monoclonal Antibodies
 Directed against a single epitope (determinant)
e.g. Most CD markers
Results in greater diagnostic accuracy
Results in less background and cleaner slides
 In vivo and in vitro methods of production
Ascites
Cell culture supernatant (90%)
Bioreactor
 Culture can be maintained indefinitely
Greater consistency from lot-to-lot

36. Monoclonal Antibodies:
Myeloma cell lines
 Requires a fusion partner (immortal cell line)
 Usually a Myeloma cell line grown in culture of that
particular species
 Most commonly used species
 Mouse
 Rat
 Rabbit (until recently only mouse and rat myelomas available)
Reference: Rabbit Monoclonal Antibodies: Generating a Fusion Partner to Produce
Rabbit-Rabbit HybridomasH Spieker-Polet, P Sethupathi, P Yam, and KL KnightProc.
Natl. Acad. Sci. USA. 1995 September; 92(20): 9348 9352

37. Monoclonal Supernatant
 Produced by
Immunizing (generating an immune
reaction) in an animal
Collecting the B-cells
Fusing the B-cells with myeloma cells
Growing cells in culture
Collecting the secreted antibodies in
the culture fluid

38. Monoclonal Antibodies
+
Human Antigen Inject into animal
serum containing several
different antibodies
Induction of immune
reaction with
production of antibody

39. Harvest spleen cellsLymphocytic proliferation
Immune reaction generates
proliferation of activated cells
Monoclonal Antibodies

40. Fuse cells with
myeloma cell
line and
grow in culture
+
+
+
+
Monoclonal Antibodies

41. anti- epitope A
anti- epitope C
anti- epitope B
anti- epitope D
Individual clones grown
separately
Each colony is
isolated into a
separate growing
vessel
Monoclonal Antibodies

42. Monoclonal Antibodies
anti- epitope D
anti- epitope A
anti- epitope C
anti- epitope B
Test clones: The different clones are specific for different epitopes
When a clone has been established
a manufacturing method must be
Selected:
•In Vivo – Ascites
•In Vitro - Bioreactor

43. Monoclonal Antibodies
Purify and package
Each selected clone can be
mass produced by the
chosen manufacturing
method

44. Monoclonal Ascites
 Ascites
 Definition: an abnormal accumulation of fluid in the abdomen
 Produced in Vivo by:
 Immunizing (generating an immune reaction) in an animal
 Collecting the B-cells
 Fusing the B-cells myeloma cells Hybridoma
 Growing cells in animals (usually rats)
 Collecting the secreted antibodies in the "tumor" fluid

45. Monoclonal Antibodies-Ascities
anti- epitope A
anti- epitope D
anti- epitope C
anti- epitope B
Different clones
are specific for
different epitopes
Inject into
animal
Collect Ascites
secreted by tumor
Purify &
Package
Generate
Tumor

46. Monoclonal Antibodies- Ascites
 Advantages of the Ascites method
 Produces high concentrations of monoclonal antibody
does not require further concentration
 Avoids effects of contaminants in in vitro batch-culture fluid
when comparable quantities of monoclonal antibodies are
used
 Avoids the need to teach cell culture technique
 Disadvantages of ascites method
 Animals must be monitored daily
 In vivo methods can contain animal proteins and other
contaminants that must be purified
 Can be expensive
 Can cause pain or distress to animals used
“Monoclonal Antibody Production,” Report of Committee on Methods of Producing Monoclonal Antibodies,
Institute for Laboratory Animal Research, National Research Council, 1999

47. Monoclonal Antibodies: In
Vitro Method of Production-
Bioreactor
 Each cartridge is inoculated with 20 million
cells
 A pump system provides a continuous
flow of fresh media to the cells trapped
within the hollow fiber cartridge
 Secreted proteins from cells cultured
within the hollow-fiber matrix are retained
within the cartridge
 Five to ten days after the initial inoculation
with hybridoma cells, up to 10 mls of
antibody rich medium is removed from the
cartridge and fresh media is injected
 Process can be repeated five times per
week

48. In Vitro Method of
Production
Bioreactor
 Non-animal alternative to the large scale production of monoclonal
antibodies
 The antibody concentration from bioreactor fluid is comparable to
ascites fluid
 Four weeks production can provide an antibody yield comparable
to the yield from 32 ascites mice.
 Unlike ascites fluid, the bioreactor fluid is free from the
contaminating mouse proteins.
 Additionally, this system provides an ideal alternative for antibody
production from cell lines that do not produce ascites in mice.

49. Rabbit Monoclonal
Antibodies
 Rabbits recognize antigens and epitopes that are
not immunogenic in mice or rats
 Were previously not possible due to lack of fusion
partner
 Plasmacytoma cell line that could be used as a
fusion partner was generated from transgenic
rabbits
 Stable hybridomas now available

50. Rabbit Monoclonal
Antibodies
 Higher Affinity
 Rabbit anti-sera recognize more epitopes
than mouse sera
 Higher Specificity
 Higher Sensitivity
 Better Development Success
 Stable hybridomas
 Because of the size of the rabbit spleen,
more fusion experiments can be performed,
making it a feasible task to screen
hybridoma at large scale

51. Rabbit Monoclonal
Antibodies
+Human Antigen Inject into animal
Generate
immune
reaction
Harvest
spleen cells
+
+
+
+
+
+
+
+
Select best clone and
grow in culture
Isolate antigen specific B-cell and
fuse with plasmacytoma fusion
partner

52. Antibody Cocktails
 Usually made up of more than one monoclonal antibody
 More sensitive than single monoclonal but more
specific than polyclonals
 Can detect multiple epitopes
 Can select the epitopes
 More effective at screening for certain proteins in
various cell types
 Are often used in combinations that are complementary or
additive
 More expensive
 Requires many clones to achieve the sensitivity of
polyclonal antibodies

53. Antibody Formats
 Antibodies may be available in
different formats or presentations
Concentrates
Predilutes
Lyophilized

54. Antibody Formats
Concentrates
 Usually sold in 1 ml sizes or smaller
 Need to be diluted using a diluent that can
either be made or purchased.
 Antibody performance can depend on the
diluent of choice.
 Buffer component
 Protein component
 Preservative
 Dilution should be optimized
 Suggested working concentration is only
a starting point

55. Antibody Formats
Predilutes
 Sold "ready-to-use"
 Usually in bottles of 5-6 mls.
 May need “tweaking” to work in your
lab
 Overall performance may depend on
Detection method
Detection source
Pretreatment method
Tissue fixation

56. Antibody Selection
 Based on:
Clone or antibody properties such as
• Specificity, sensitivity, stability
Publications
Application (clinical utility)
Pathologist preference
Peer recommendations
Vendor
 Finding information on antibodies can be
challenging

57. Specification Sheets
 Depending on Regulatory classification of
the antibody, Specification Sheet may
provide information on
 Antibody specificity
 Specie
 Immunogen
 Clone
 Isotype
 Concentration
 Ig concentration
 Suggested Working concentration
 Intended Use
 Clinical utility: Diagnostic vs.
Prognostic
 Storage Conditions
 Temperature and stability
 Suggested Protocol
 Pretreatments
 Incubation
 Detection
 Regulatory status
 PMA
 IVD
 ASR
 Research

58. Specification Sheet: Zymed’s
CD20

59. Specification Sheet: Zymed’s
CD20

60. Specification Sheet: Zymed’s
CD20

61. Diluents
 Can make a significant difference to
antibody performance
 Antibodies from one vendor may not
be stable in diluent from another
vendor
Stability
Background

62. Diluents
Composition of a Antibody Diluent:
 Buffer
 TBS (pH is critical - must be pH7.6)
 PBS (pH 7.2-7.4)
 Protein stabilizer
 FCS
 Normal serum (10-20*%)
• high concentration of serum in the ab diluent can alter the
pH
 BSA (0.1- 1% BSA in PBS)
 Casein .03% in PBS
 Preservation
 0.002 - 0.1% NaAzide
 Kathon (Rohm & Haas)
 Sterilization
 Filtration

63. Basic IHC techniques:
Fixatives
Common Fixatives IHC techniques are
divided into two groups
- Coagulant fixatives such as ethanol, and
cross linking fixatives, such as
formaldehyde.
- Both can cause changes in the steric
configuration of proteins, that can mask
antigenic sites (epitopes) and adversly
affect binding with antibody

64. Basic IHC techniques:
Fixatives
 Formalin has been the standard fixative of use with the
most advantages revealed in the course of history:
1. Good preservation of morphology for a variety of tissues
2. Formalin is an economic chemical
3. Formalin fixation acts to sterilize tissue, especially
containing viruses
4. Antigens in Carbohydrates are better preserved 111
5. Through cross linking of protein, antigenicity is preserved in
situ, therefore; avoiding leaching out of proteins that may
diffuse in alcohol or methanol.

65. Basic IHC techniques:
Pretreatments Heat
Unmasking
 Uses high heat combined with a liquid (usually a buffer)
to undo effects of fixation
 Microwave to heat the liquid for the purpose of
unmasking antigens
 Boiling - a beaker over a hot plate etc
 Steamer
 Stove top pressure cooker
 Microwave pressure cooker
 Electric pressure cooker
 Autoclave
 Waterbath
 There are also many different buffers, such as
 Citrate buffer, citrate buffer/urea, citrate buffer/EDTA
 EDTA, EDTA/urea
 TRIS
 Glycine
 etc. etc. etc.

66. Pretreatments
Heat Unmasking
Antibody binding site
on antigen. Fixation causes bonds
to form across
portions of the protein
(cross-linking).
Antibody cannot bind
to site.
Unmasking breaks
bonds so that binding
site is available for
antibody binding.

67. AR Pitfalls
 No testing for pH stability in AR buffers
 Non testing of heating system for AR
 Not familiarizing yourself with the following prior to performing
AR-IHC staining:
1. The cellular localization of the antigen base
2. Specificity of the primary antibody
3. Previous IHC staining results from literature, especially from an
experienced laboratory.
4. Any adverse influence on the antigen from tissue fixation,
processing, the necessity of any pretreatment procedures (heat-
induced AR)
5. Not reading the package insert! Information regarding reagents,
antibody clone, detection systems, manufacturer, recommended
concentration, etc.

68. Pretreatments: Heat Unmasking
 Heat Unmasking:
- Heat unmasking is affected by:
Type of buffer
pH of buffer
Exposure time in solution
Cool down times
Pressure
Fixation
- All antigens do not respond equally to the same unmasking
conditions
- Antigen unmasking solutions should not be reused

69. Pretreatments: Heat Unmasking
 Heat Unmasking:
- Can increase non-specific staining (background) by
exposing previously cross-linked endogenous substances
- Needs to be optimized independently for most labs
- Solutions used for heat unmasking should never be reused
because:
fixative can be dissolved out into the solution
can become saturated with fixative, so that it fixes
instead of unmasking
pH can drift

70. Basic IHC techniques
Pretreatments: Blockers and
Enzymes
Attendees will gain a basic knowledge of:
 Endogenous Peroxidase
 Endogenous Biotin
 Proteases (Enzymes)
And pitfalls to avoid

71. Basic IHC techniques
Pretreatments: Endogenous
Peroxidase
 Endogenous Peroxidase Facts
1. Peroxidase molecules naturally occur, ie, endogenous in bloody
tissue sections fixed in paraffin. These tissue sections will react
in the substrate-chromogen step in the detection procedure.
Red blood cells will stain when exposed to diaminobenzidine
and hydrogen peroxide because of their endogenous peroxidase
content.
2. Poor fixation contributes to endogenous peroxidase activity
because peroxidase can leach out of the red blood cells into the
surrounding tissue increasing the background staining.
3. Note: frozen tissue sections lose their endogenous peroxidase in
their red blood cells due to cell lyses in the freezing process.

72. Basic IHC techniques:
Pretreatments: Endogenous Biotin
Highly charged molecules exist within any given tissue as
normal components. These molecules may not be the
target antigen of a given immunohistochemical protocol.
During application of a primary antibody, if the target
antigen is present, the primary antibody will bind to it,
resulting in a immunospecific reaction. However, in
circumstances where the tissue has not been adequately
blocked the primary antibody also may combine with non-
target sites, resulting in a non-immunospecific reaction.
If this happens, the secondary antibody also will bind,
leading to background staining.

73. Blockers
 Agents that are used to prevent or reduce false-positive or non-
specific staining
 Staining that is not related to primary antibody binding to the
antigen
 Also called "background" staining
 Non-specific staining has many sources and can vary with
 Tissue
 Fixation
 Pretreatment
 Antibody
 Staining protocol
 Detection system
 Selection and application of blocker depends on the source of
non-specific staining

74. Blockers
 Potential sources of non-specific staining
 Protein interactions
• General binding of proteins to each other due to compatible
structure and charges
 Endogenous enzymes and proteins with enzymatic activity
• Peroxidase
• Hemoglobin
• Alkaline phosphatase
 Endogenous Biotin
 Interstitial Ig
 Cross-Reactivity of Primary antibody
 Antigen Diffusion( improper, inadequate or delayed fixation
which may allow the antigen of interest to diffuse from the site
of synthesis or storage and disperse throughout the tissue)
 Heat unmasking can increase the incidence of all of the above

75. Blockers
Protein Blockers
Protein structures on the surface of
the tissue or cells bind the antibody
non-specifically
Addition of nonspecific protein, prior
to application of primary antibody,
blocks non-specific sites.
B
B
B
B
B
B
B
B
B
B
B
B

76. Blockers
Biotin Blockers
B
B
B
B
B
B
B
B B
BB
AA
A A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
Endogenous biotin in the tissue will bind to the avidin/enzyme
complex. This will produce color reactions at the site of
binding.
Endogenous biotin is blocked by:
1. Addition of free avidin which blocks biotin's one binding
site.
2. Addition of free biotin which will block all four avidin
binding sites

77. Inhibition of Endogenous
Peroxidase Mechanism
 The mixture of Methanol and hydrogen peroxide
quenches endogenous peroxidase without altering
the subsequent antibody reaction.
 Because of the phenomenon of Substrate
Inhibition, hydrogen peroxide can act in dual roles,
as inhibitor and substrate.
 Remember that in the detection system, hydrogen
peroxide is the substrate that acts on the
peroxidase enzyme to form a colored product.

78. Inhibition of Endogenous
Peroxidase Pitfalls to avoid
 Inadequate concentration of hydrogen
peroxide to insufficient to irreversibly inhibit
the endogenous enzyme. A 100 fold
concentration of 0.03% is needed to block
endogenous activity.

79. Blockers
Blocker Type of blocker
H202, sodium azide Peroxidase
Hydrolyzed casein Protein
Levamisole Alkaline Phosphatase
BSA Protein
Free avidin Biotin
Free biotin Avidin
Hydrolyzed casein Protein
Goat Ig Protein

80. Endogenous Biotin: Pitfalls
to avoid
 Must have both blockers avidin and biotin.
 Must place avidin blocker on tissue prior to
biotin blocker.
If the end IHC staining results in brown
staining covering the entire surface of the
tissue, the most probable cause is that
biotin blocker was not added after avidin
blocker and the detection system linked to
the avidin within the tissue.

81. Basic IHC techniques
Pretreatments: Proteases
 Proteases
1. Enzymatic epitope retrieval is defined as a method used to relax the
rigidity of the protein structure that results from the cross linkages of
formalin fixation.
2. Proteolytic enzymes are used in an attempt to restore the
immunodominant structure in the epitope of interest. This method
makes an epitope available to associate with its antibody.
3. Proteolytic enzymes are thought to cleave proteins at specific
locations depending on the specificity of the enzyme. If cleavage
points are in proximity to a cross-link, then the resulting effect is a
relaxation of the rigid protein structure facilitating contact between
the primary antibody and the corresponding antigenic determinant.
Ancillary Methods in Immunohistochemistry, Immunhistochemical Staining
Methods, 4th
Edition,2006,71.

82. Proteases
 Each enzyme responds to a specific amino acid
sequence. Since the specific cleavage sites are
usually unpredictable, the procedure is not always
successful and sometimes results in the loss of
certain epitopes.
 Typically enzymatic digestion doesn’t affect
epitopes with high carbohydrate content. However,
it can be appropriate for glycoprotein-rich targets,
such as the epitope for glucagon immunoreactivity
in certain tumors.

83. Proteases
 Conditions and enzymes used for unmasking could
be different for each antigen.
 The optimal temperature for most proteolytic
enzymes used for IHC is about 37 C
 Lower temperatures are possible and in some cases
are preferable because they allow a greater degree
of control over the digestive process.

84. Pretreatments: Protease
Enzyme Unmasking
 The most commonly used enzymes are
 Proteases
 Pronase
 Proteinase K
 Pepsin
 Trypsin
 Ficin
 Each enzyme has a unique enzymatic activity
level
 Activity level varies with:
• Concentration
• pH
• Temperature

85. Pretreatments: Proteases
Pitfalls to avoid
 Needs to be controlled very
carefully
Can be harsh
 Too little, too much or the wrong
one can
Prevent staining altogether
Result in inappropriate staining

86. Pretreatments
 Pretreatment allows staining of paraffin embedded
tissue with many antibodies over a wide variety of
fixation
 Improves clinical utility of many primary antibodies
 Numerous methods are available and they all have
different advantages and disadvantages
 There is no universal pretreatment and no industry wide
standardization
 Pretreatments do not just unmask epitopes, but also
expose potential sources of background which may
require blocking

87. Second Half !
Break Time