Inside out of IHC. DR SHANKAR BASTAKOTI PATHOLOGIST BP KOIRALA CANCER HOSPITAL

1. IMMUNOHISTOCHEMISTRY
Dr. Shankar Bastakoti
JR, Pathology
3rd year

2. • Technique for identifying antigens in tissue (‘histo’) by
means of antigen-antibody interactions (‘immuno-
chemistry’)
• The advent of diagnostic immunohistochemistry has
made such an impact so known as ‘brown revolution’
and as ‘magic markers’.

3. History
1942
• Coons, Creech, Jones, Berliner: Developed indirect immunofluorescence
method to demonstrate pneumococcal antigen in tissue
1966
• Graham & Karnovsky, Developed enzyme tagging method [horseradish
peroxidase]
1967
• Nakane & Pierce, Developed enzyme-labelled antibody technique
[immunoperoxidase]
1970
• Sternberger, Developed unlabelled antibody method [peroxidase-
antiperoxidase (PAP) technique]
1974
• Heitzman & Richards, Developed another unlabelled antibody method
[avidin-antibiotin complex (ABC) technique]
1996
• Dako developed polymeric techniques like EPOS and more recently
EnVision detection system

4. Principle
• Immunohistochemistry : detecting the presence of specific
proteins (antigen) in cells or tissues
Existence and distribution of an antigen detected micro
chemically is made manifest macro chemically
• Sharp localization of target components in cell and tissue is
based on a satisfactory signal-to-noise ratio
Amplifying the signal while reducing the nonspecific background
staining(noise) is the major strategy
Immunohistochemistry is the localization of antigens or
proteins in tissue sections by the use of labeled antibodies
as specific reagents through antigen-antibody
interactions that are visualized by a marker such as
fluorescent dye, enzymes etc.

5. Structure of Protein (antigen)
• Antigen is a molecule that
induces the formation of
an antibody.
• Bears one or more
antibody binding sites.
• Epitopes/ antigenic
determinant groups,
composed of small number
of aminoacids or
monosaccharide units form
highly specific topographic
sites for antibody binding.
Antibody structure - monomer
IgG is the commonest and most
frequently used antibody for IHC

6. Antigen – Antibody binding
Binding
• AA side chains of variable
domain of antibody forms
a cavity, geometrically and
chemically complementary
to a single type of antigen
epitope
• The associated antibody
and antigen are held
together by a combination
of hydrogen bonds,
electrostatic forces and Van
der Waals’ forces
Factors in an Ag-Ab reaction
• Affinity: 3D fit of antibody to
the antigen epitope
• Measure of the binding strength
• Avidity: Multivalency of the
antibody
• Multiple antibodies reacting
with different epitopes of the
same antigen are less likely to
be removed by washing
• Functional combining strength
• Specificity
• Sensitivity

7. Primary antibodies
Polyclonal antibodies
• Obtained by immunising an animal with a purified specific immunogen
(antigen of interest) and harvested by bleeding to obtain immunoglobulin
rich serum
• Mixture of antibodies to different epitopes .
• Also contain antibodies to impurities on the immunogen (usually of low titre
and affinity)
• May cross react with other molecules
• SENSTIVE BUT LESS SPECIFIC – more background staining
Monoclonal antibodies
• Hybridoma technique by Kohler and Milstein (1975)
• Constant reliable production of one pure antibody with one epitope
specificity
• Cross reactivity to other molecules can be eliminated by screening and
careful selection of hybrids
• Dramatically increased the number of antibodies available for IHC,
particularly in paraffin embedded sections
Lectins
• Plant or animal proteins that bind tissue carbohydrates with high specificity

8. Direct method
• Primary antibody is
conjugated directly to
the label
• Little signal amplification
and lower sensitivity
• Mainly used in
demonstration of Ig and
complement in frozen
sections of skin and renal
biopsies
Indirect method
• Primary antibody is
unlabelled
• Secondary antibody directed
against the immunoglobulin
is conjugated with the label
• More signal amplification and
higher sensitivity
• Versatile
• Same secondary antibody can be
used for a multiple primary
antibodies of the same animal
species

9. LABELS
• Enzyme labels
• Enzyme incubated with chromogen produces a stable coloured
reaction end product
• Enzymes:
• MC used - Horse Radish peroxidase with chromogen DAB
• Calf intestinal alkaline phosphatase
• Glucose oxidase
• Bacterial derived B-D-galactosidase
• Colloidal metal labels
• Colloidal gold alone or with silver precipitation to amplify visibility
and enhanced using polarised incidental light.
• Fluorescent labels
• Radiolabels
• Although requires auto radiographic facilities, it can quantify reaction
• Limitation of availability of internally labeled antibodies

10. Enzyme labels – Horse Radish
peroxidase
• Why HRP?
• Small size – does not hinder binding of antibody to adjacent sites
• Easily obtainable in purified form
• Stable
• Endogenous activity is easily quenched.
• Endogenous activity?
• Endogenous peroxidase activity is present in neutrophils and myeloid
cells
• Blocking procedures, most popular is hydrogen peroxide-methanol
method
• Chromogen
• MC – 3,3’-diaminobenzidene tetrahydrochloride – dark brown
reaction product
• Initially reported to be a potential carcinogen, the risk is now thought
to be low.

11. Endogenous peroxidase blocking
• Most commonly used method:
• Incubation in absolute methanol containing 0.5%
hydrogen peroxide for 10 minutes in room
temperature
• Produce almost complete abolition without affecting
immunoreactivity
• Can be done before or after primary antibody
incubation. Generally blocking after primary antibody
is recommended as it may interfere with binding of
primary antibody to antigen

12. Immunohistochemical methods
Direct technique
• Traditional direct technique:
• Hardly used except in skin and kidney using more commonly fluorochrome
conjugates (IF)
• Quick and easy
• BUT
• Helpful only with monoclonal antibody
• For each different antigen it is necessary to conjugate each primary antibody separately
• High concentration of antibody required
• New direct technique:
• EPOS – Enhanced Polymer One Step staining
• A large number of primary antibody molecules and peroxidase enzymes are
attached to a dextan ‘polymer’ blackbone
• Signal amplification is increased and more sensitivity
• Time duration reduced
• However, limited number of such primary antibodies available
FITC
FITCFITC
FITC
FITC
FITC
FITC
FITC
FITC

13. Immunohistochemical methods
Indirect technique
• Two Step indirect technique
• Polymer chain two step indirect technique
• Unlabeled antibody-enzyme complex techniques
HRP
HRP
HRP
HRP
HRP
HRP
HRP

14. Polymer chain two step technique
• Unconjugated primary
antibody followed by a
secondary antibody
conjugated to a labeled
polymer dextran chain
• 70 molecules of the label
(HRP enzyme) and 10
molecules of secondary
antibody
• Polymer may contain both
anti-mouse and anti-rabbit
IgG and therefore can be
used for both mouse and
rabbit primary antibodies
• Biotin free
• Quick, reliable and
reproducible method

15. Unlabelled antibody-enzyme complex
techniques (PAP and APAAP)
• PAP (peroxidase-
antiperoxidase) - three step
• Unconjugated primary antibody
• Unconjugated second layer of
bridging antibody which has
binding sites for both primary
antibody and third layer
complex
• Third layer of PAP complex – 3
peroxidase molecules and 2
anti-peroxidase molecules
• APAAP technique
• Alkaline phosphatase
antibodies raised in mouse can
be used by the same principle
• These methods have been
largely replaced by ABC and
polymer methods

16. Immunohistochemical methods
(Strept)avidin-biotin technique (ABC)
• Most widely used methodology in diagnostic IHC along with indirect
polymer techniques
• Three step technique
• These methods rely on the marked affinity of the basic glycoprotein avidin
for the small water soluble vitamin biotin (Vit H)
• Disadvantages of avidin:
• High isoelectric pH of 10; therefore it has + charge at neutral pH leading to non specific binding with
negative charged structures such as nuclei
• Being a glycoprotein, it can react with carbohydrate moiety of lectins leading to non specific reactions
• Substitution of streptavidin overcomes these problems
• Streptavidin isolated from bacterium Streptomyces avidini
• 4 high affinity binding sites for Biotin
• Streptavidin contains no carbohydrate which can bind nonspecifically to lectin like substrate
• Isoelectric point is closed to neutrality
• Highly stable reagent
• Biotin
• Biotin is easily conjugated to antibodies and enzymes
• Up to 150 biotin molecules can be attached to one antibody molecule

17. (Strept)avidin-biotin technique (ABC)
• The ABC technique can employ either enzyme bound directly
to the streptavidin
• Alternatively, both secondary antibody and the enzyme can be
biotin labelled and streptavidin acts as a bridge between them
Biotin
Biotin
Biotin
Biotin
Biotin
Biotin
Biotin
Biotin
BiotinA vidin
HRP
A vidin
HRP A vidin
HRP
A vidin
HRP
A vidin
HRP
A vidin
HRP
A vidin
HRP
A vidin
HRP

18. (Strept)avidin-biotin technique (ABC)
Endogenous biotin blocking
• Tissues rich in endogenous biotin such as liver and kidney will
require the use of avidin/biotin block before applying primary
antibody
• Block the slides for 15 minutes in Avidin buffer.
• Avidin Buffer: 0.001% Avidin in 0.1M PBS, pH 7
• Wash in buffered Tris saline after blocking

19. STEPS OF IHC
Slide 3 of 23
Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Secondary antibody
Primary antibody
Microscopy Observation
Blocking background staining
Chromogen Substrate
Counter stain
Mounting

20. Step 1: Deparaffinization
• Generally polylysine (0.1% solution) coated or charged slides are
used to ensure that the sections are adherent to the slide
• This step removes the paraffin still remaining on slide, which
would otherwise interfere with the ability of the antibodies to
detect the proteins of interest.
Paraffin Proteins

21. Step 2: Antigen Retrieval
• Fixation modifies the tertiary structure of proteins(Ags) -
many times making them undetected by specific Abs
• Cross-linking fixatives like formalin lead to formation of
methylene bridges which cross-link proteins and therefore
mask antigenic sites
• Cross-linking the irrelevant protein to the peptide epitopes
sterically blocked antibodies from binding
• The epitopes for clinical antibodies require only primary
protein structure
• Antigen retrieval dissociates irrelevant proteins and restores
immunoreactivity by probably denaturing the secondary and
tertiary structure of the irrelevant protein.

22. Methods of Antigen Retrieval
•Microwave Oven, Pressure Cooker and Steamer are the most
commonly used heating devices. Other devices also include the use
of autoclave and water bath
•The heating length of 20 minutes appears to be the most
satisfactory and the cooling usually takes about 20 minutes
Heat Induced Epitope
Retrieval (HIER)-
•Proteinase k, trypsin, chymotrypsin, pepsin, pronase and various
other proteases
Proteolytic Induced
Epitope Retrieval
(PIER)-
•Alternative approach to unmask antigens if other methods did not
work
•Especially useful when performing double or triple labeling of two
or more antigens simultaneously
Combination of Heat
Mediated and
Proteolytic Enzyme
Method-

23. Heat induced epitope retrieval
Two factors are important:
• PH value of AR solution
• Most antigens – no significant variation in pH value ranging from 1.0-10.0
• 0.01 M Citrate buffer of pH 6.0 is the most popularly used retrieval solution
• The TRIS-EDTA of pH 9.0 and 0.1mM EDTA of pH 8.0 are second most used
retrieval solutions
• 0.01 M TRIS-HCl, pH 1 or 10, was slightly superior to citrate buffer of pH 6.0 and
gave the best overall results .
• Heating temp and heating time-
• The relationship between the temperature and time is inverse
• Temperature below 80*c – not significantly beneficial

24. A New Method for Unmasking
Antigens
• LAB Solution (liberating antibody binding)
• Thiocyanic acid, compd. with guanidine 1 (1:1) + Water
• An effective method for routine unmasking of antigens for
IHC staining based on simple protein chemistry
• Allows antigen unmasking in less time and at room
temperature or much lower temperatures than other methods
• Can be used on automated stainers whether they have
heating capabilities or not .

25. Step 3: Blocking
• Major causes of background staining
• Hydrophobic and ionic interactions
• Hydrophobic interactions are the result of cross linking of amino
acids both within and between adjacent protein molecules due to
aldehyde fixation
• Collagen, epithelium and adipocytes give rise to background staining due
to hydrophobic interactions
• Non specific staining is most commonly produced because the
primary antibody is attracted non-immunologically to highly charged
groups present on connective tissue elements.
• Endogenous enzyme activity
• Peroxidase blocking
• Biotin blocking

26. Serum Blocking
• Serum blocking is an important step. The presence of serum
proteins keeps the antibodies from binding non-specifically to the
slide and reduces non specific background staining. Do not wash
after serum blocking.
• Endogenous enzyme blocking for peroxidase x10 minutes
• Biotin block, if necessary
Serum
Proteins

27. Step 4: Washing
• After almost every step, except serum blocking, a wash should
be performed. This will remove unwanted proteins or
chemicals
• Wash in Tris buffered saline
Unbound
Proteins
WASH

28. Step 5: Primary antibody incubation
• For optimum staining, primary antibody is to be used at
correct dilution. Too high and too low concentrations may
cause false negative results
• Generally, around 100 µl is the quantity of diluted primary
antibody required for a one section
• A moist chamber
• Time for incubation depends on the individual antibody
varying from one hour to overnight incubation
• Wash in TRIS buffered saline

29. Step 6: Secondary antibody
incubation
• In a standard 3 step ABC
protocol
• Incubate with secondary reagent
(biotinylated goat anti-rabbit
immunoglobulins) followed by
buffer rinse
• Incubate slides with ABC complex
conjugated with HRP followed by
buffer rinse
• In a two step HRP polymer
method
• HRP polymer secondary antibody
incubated in a single step

30. Step 7: Detection of immune
reaction by chromogen
• Addition of DAB reagent or other chromogen for 5- 15
minutes
• Stop reaction by bringing sections to deionized water

31. Step 8: Counter Staining
• Counterstain with Mayer’s hematoxylin (dark purple)
• Dehydrate and coverslip

32. What we do here!!! What Aiimsians do!!!
Tissue sections 3 to 4 micro thickness
on APES (3-aminopropyl triethoxy
silane) coated slides.
Tissue sections 3 to 4 micro thickness
on APES coated slides.
Slides are left in hot plate for few
minutes and later dipped in xylene (2
change, 5 mins each).
Slides are left in hot plate for few
minutes and later dipped in xylene (2
change, 5 mins each).
Slides are then rehydrated through
acetone (two change, 5 mins each),
alcohol for 5 mins and running tap
water for 5 mins.
Slides are then rehydrated through
acetone (1 change, 5 mins ), alcohol for
5 mins and running tap water and
distilled water for 5 mins.
Slides are not allowed to dry at any
point.
Slides are not allowed to dry at any
point.
Antigen retrieval is performed by
placing slides in citrate buffer in
pressure cooker for 15 mins.
Antigen retrieval by placing slides in
citrate buffer in microwave oven at
100⁰ C for 30mins (100 C = 900 W in
microwave)

33. Slides are cooled at room temperature
and next steps of staining are
performed in moist chamber
Slides are cooled at room temperature
and next steps of staining were
performed in moist chamber.
Slides are washed with wash buffer
(Tris buffer solution).
The slides are washed with running
tap water.
Slides are incubated in Hydrogen
peroxide block (3% hydrogen peroxide
in methanol) for 10 mins.
Slides are incubated in Hydrogen
peroxide block (4% hydrogen peroxide)
for30 mins.
Slides are washed with Tris buffer (4
changes, 5 mins each).
Slides are washed with running tap
water and distilled water and 1 change
of Tris buffer.
The primary antibody is added and
kept overnight in freeze.
Ultravision protein block for 5 mins at
room temperature (100 ml of Tris
buffer with 0.5 ml of protein block)

34. Washed with wash buffer and super
enhancer (secondary antibody, Ultravision
One HRP Polymer) is applied for 30 mins
and washed with wash buffer 3 times.
Washed with Tris buffer (4 changes, 5 mins
each).Marked with pap pen (paraffin pen)
The primary are added and incubated at
room temperature for 2 hrs.
DAB added to slide for 10-15 mins and
washed with Tris buffer.
Slides were washed with wash buffer ( 4
changes).
Primary antibody amplifier quanto is added
and kept for 15 mins
The slides are dipped in Harris hematoxylin
for 30 secs and washed in running tap
water for 5 mins.
HRP polymer quanto is added and kept for
30 mins.
The slides are washed with tris buffer (4
changes).
DAB added to slide for 3 mins and washed
with running water.
Dipped in hematoxylin for 30 secs and
washed in running tap water for 5 mins.
The slides are air dried and mounted with
diphenylene phthalate xylene
The slides are air dried and mounted with
DPX.

37. Trouble shooting
• False negative
• Incomplete deparaffinization
• Incorrect retrieval solution
• Inadequate heat induced epitope retrieval temperature
• Extreme digestion using enzyme retreival
• Wrong antibody concentration
• Incorrect chromogen
• Wrong positive control
• Human or machine error

38. Trouble shooting
• False positive staining
• Poor fixation
• Poor technical quality of sections
• Variation in pH of retrieval solution
• Endogenous enzyme activity and inadequate blocking
• Species cross reactivity
• Tissue drying (edge artifacts)
• Chromogen precipitation and streaking
• Automation error usually incomplete rinsing

39. ABSENT STAINING OR WEAK
STAINING
PROBLEM SOLUTION
INADEQUATE FIXATION Avoid delay of fixation( >30 min) or
overfixation (>48 hrs)
INCOMPLETE DEHYDRATION DURING
PROCESSING
Perform regular reagent changes(i.e. Alcohol)
PARAFFIN TOO HOT (heat sensitive epitopes
lost)
Monitor temp of paraffin(<56⁰C)
PROLONGED OR EXCESSIVE HEATING Optimize AR time
ANTIBODY TOO DILUTE Check Ab titration; increase conc ; lenghten
incubation time; increase temp of Rxn
DRYING OUT OF TISSUE DURING PROCESSING Keep specimen moist; use humidity chamber
INSUFFICIENT INCUBATION TIME Lengthen incubation time; add heat; increase
conc of Ab
CHROMOGEN NOT WORKING Add chromogen to labelling solution and
monitor for change in color
REAGENT NOT WORKING Check expiry, pH and compatibility with other
reagents

40. BACKGROUND STAINING
PROBLEM SOLUTION
NONSPECIFIC PROTEIN BINDING Use nonimmune serum from same
animal species as sec Ab; add salt to
buffer
POORLY FIXED OR NECROTIC TISSUE Make sure tissue is properly fixed;
avoid sampling of necrotic areas
THICK PREPARATION Cut sections at 3 to 5 mm
INAPPROPRIATELY CONC Ab Check titration; decrease conc;
decrease incubation time; decrease
temp of Rxn
ENDOGENOUS BIOTIN Block with avidin
INCOMPLETE RINSING OF SLIDES Follow the protocol for proper slide
rinsing
CHROMOGEN STAINING TOO INTENSE Monitor timing of chromogen
substrate Rxn; filter the chromogen;
decrease the chromogen conc.

41. Common applications of
Immunohistochemistry
• Characterization of Poorly differentiated malignancy
• Differentiating in-situ lesion from invasive malignancy
• Metastatic tumour work up
• Determination of viral etiology
• Prognostic and therapeutic value

42. Interpretation of Immunohistology
Signal pattern: Sub-cellular localization
Four broad categories
1. Membranous pattern
2. Nuclear pattern
3. Cytoplasmic pattern
4. Nuclear-cytoplasmic

43. THANK YOU