Important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment. pathogenesis, diagnosis, and treatment of diseases are discovered.It is performed without destruction of histologic architecture. It can be used as an effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of definitive diagnosis and also gives an insight into tumor histopathogenesis and prognosis.

1. IMMUNOHISTO
CHEMISTRY
-Dr. DEVIKA.K.V
POSTGRADUATE
DEPT OF ORAL PATHOLOGY

2. SYNOPSIS
INTRODUCTION
IMMUNOHISTOCHEMISTRY
ANTIGEN AND ANTIBODIES
PRODUCTION OF PRIMARY REAGENTS
ENZYME LABELS
IMMUNOHISTOCHEMICAL TECHNIQUES
UNMASKING OF ANTIGEN SITES
DETECTION OF LOW LEVELS OF ANTIGEN
BLOCKING OF ENDOGENOUS ENZYMES
IMMUNOHISTOCHEMISTRY IN PRACTISE
IMMUNOHISTOCHEMISTRY QUALITY CONTROL
CONCLUSION
REFERENCES

3. INTRODUCTION
• important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical
research including exploration of biomarkers, as IHC allows confirmation of target molecule
expressions in the context of microenvironment.
• pathogenesis, diagnosis, and treatment of diseases are discovered.
• performed without destruction of histologic architecture.
• effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of
definitive diagnosis.
• insight into tumor histopathogenesis and prognosis.

4. Immunohistochemistry
• Immunohistochemistry is the localization in tissues of an (known) antigen by means of antibodies
directed towards that (specific) antigen.
• Ab-Ag complex is visualised using a marker.
• Immunocytochemistry refers to localization in isolated cells or labelling directed to cell specific
compartment (e.g. the cell membrane, Golgi or lysosomes, etc.).

5. ANTIGEN
Molecules that interact specifically with the products of immune response generated by
an immunogen, that is, with antibodies, B cell receptors (BCR) and/or T cell receptors
(TCR).
The two attributes of antigenicity are:
Immunogenicity
Immunological activity
Based on the ability to carry out these two functions, antigens may be classified further
into
A complete antigen- can induce antibody formation
Haptens- are substances that are incapable of inducing antibody formation

6. EPITOPE
The smallest unit of antigenecity
It contains four or five amino acids or monosaccharide residues that possess a specific
chemical structure, electric charge and steric(spatial) configuration.
PARATOPE
The combining area on the antibody, corresponding to the epitope
Paratope + Epitope
Ab-Ag complex

7. ANTIBODY
Antibodies are glycoprotein molecule that recognise
a particular epitope on the antigen, bind
specifically to it and finally facilitate the clearance
of the antigen
Secreted by the plasma cells and are present on the
B cell membrane

8. ANTIGEN AND ANTIBODY BINDING
The amino acid side chains of the variable domain of an antibody forms a cavity which is
geometrically and chemically complimentary to a single type of antigen epitope
NON COVALENT INTERACTIONS
Hydrogen bonding
Electrostatic bonding
Vanderwals forces

9. AFFINITY
Three dimensional fit of the antibody to its specific antigen and is a measure of the
binding strength between the antigen epitope and its specific antibody combining site.
AVIDITY
Property referring to the heterogeneity of the antiserum which contains various
antibodies reacting with different epitopes of the antigen molecule.
ANTIBODY SPECIFICITY
characteristics of an antibody to bind selectively to a single epitope of an antigen
SENSITIVITY
refers to the relative amount of antigen that an immunohistochemical technique is able
to detect

10. PRODUCTION OF PRIMARY REAGENTS
POLYCLONAL ANTIBODIES
Humoral response initiated by the animal
Numerous clones of activated plasma cells are produced

11. MONOCLONAL ANTIBODY
Hybridoma
technique by
Kohler and
Milstein in
1975

12. Tissue section
Antigen retrieval
Blocking of
endogenous enzyme
Primary antibody
Secondary antibody
Chromogen substrate
Counterstain
Mounting
Microscopic observation

13. LABELS
ENZYME LABELS
• Most widely used
• Incubation with a chromogen using a standard histochemical method produces a stable,
coloured reaction end-product suitable for light microscope
HORSE RADISH PEROXIDASE
• It is small and doesnot hinder the binding of antibodies to
antigen
• Easily obtainable in purified form and less chances of
contamination
• Stable and remains unchanged during manufacture, storage
& application
• Endogenous activity is easily quenched
 3- amino-9-ethyl carbazole- red
 4-chloro-1-naphthol-dark blue
product
 α-naphthol pyronin- red purple
3,3α-diaminobenzidene
tetrahydro_x0002_chloride (DAB)-
brown

14. • Some of these chromogens produce reaction products which are soluble in xylene and
alcohol, hence these sections require aqueous mounting eg. neutral buffered glycerin
jelly
• Dry in hot air owen
• For long term storage use coverslip and resinuous mountant on top of the hardened
aqueous mountant
• Endogenous peroxidase activity is present in the number of sites, particularly neutrophil
polymorphs and other myeloid cells.
• Blocking procedures- hydrogen peroxide- methanol method
• Care should be taken with certain antigens (CD 4)- too long incubation in blocking
solution and too long in horse radish peroxidase result in staining reaction

15. • Calf intestinal alkaline phosphatase – widely used alternative enzyme tracer to horse
radish peroxidase (levamisole, 20% glacial acetic acid blocks endogenous alkaline
phosphatase)
• Glucose oxidase can be used as a tracer and it can be developed to give a navy blue
reaction
• Bacterial derived β-D-galactosidase has also been used as a tracer and can be developed
to give a turquoise blue reaction end product.
COLLOIDAL METAL LABELS
o When used alone colloidal gold conjugates
o appear pink when viewed using LM
o A silver precipitation reaction can be used to
amplify the visibility of the gold conjugates

16. IMMUNOHISTOCHEMICAL TECHNIQUES
Traditional direct technique
Two step indirect technique
Immunogold silver staining technique
(Strept) avidin-biotin technique
Hapten labelling technique
Unmasking of antigen sites
Heat mediated antigen retrieval techniques
Hapten labeling technique
Combined microwave antigen retrieval and
trypsin digestion
Detection of low levels of antigen

17. TRADITIONAL DIRECT TECHNIQUE
◦ The primary antibody is conjugated directly to the label.
◦ The conjugate may be either a fluorochrome (more commonly) or an enzyme.
◦ The labeled antibody reacts directly with the antigen in the histological or cytological
preparation.
• Quick and easy to use.
• Provides little signal amplification
• Lacks the sensitivity.

18. TWO-STEP INDIRECT TECHNIQUE
• A labeled secondary antibody directed against the immunoglobulin of the animal species
in which the primary antibody has been raised visualizes an unlabeled primary antibody.
• Horseradish peroxidase labeling is most commonly used, together with an appropriate
chromogen substrate.
• More sensitive technique because multiple secondary antibodies may react with different
antigenic sites on the primary antibody, thereby increasing the signal amplification.

19. POLYMER CHAIN TWO-STEP INDIRECT TECHNIQUE
◦ This technology uses an unconjugated primary antibody, followed by a secondary
antibody conjugated to an enzyme (horseradish peroxidase) labeled polymer (dextran)
chain.
◦ Conjugation of both anti-mouse and anti-rabbit secondary antibodies enables the same
reagent to be used for both monoclonal (rabbit and mouse) and polyclonal (rabbit)
primary antibodies.

20. Unlabeled Antibody Methods
PEROXIDASE ANTIPEROXIDASE METHOD / PAP
◦ Immune complex typically consists of 2 antibody molecules and 3 HRP molecules in the
configuration.
◦ The PAP reagent and the primary antibody must be from the same species, whereas the
bridge or linking antibody is derived from a second species and has specificity against the
primary antibody and the immunoglobulin
◦ incorporated into the PAP complex.

21. ALKALINE PHOSPHATASE–ANTIALKALINE PHOSPHATASE METHOD
/ APAAP
◦ Principle same as those described for the PAP method except that the PAP complex is
replaced with an APAAP complex.
◦ The method has had three major applications:
◦ (1)staining of tissues with high levels of endogenous
peroxidase,
◦ (2)double immunostaining in conjunction with
peroxidase,
◦ (3)staining of specific cell types that benefit from the
bright red color of alkaline phosphatase substrates

22. IMMUNOGOLD SILVER STAINING TECHNIQUE
(IGSS)
◦ Introduced by Faulk and Taylor (1971).
◦ The gold particles are enhanced by the addition of metallic
silver layers to produce a metallic silver precipitate which
overlays he colloidal gold marker.
◦ silver lactate - ion supplier
◦ hydroquinone - reducing agent.
◦ Disadvantage: Formation of fine silver deposits in the
background

24. BIOTIN-AVIDIN PROCEDURE
Biotin is linked chemically to the primary
antibody,
Produces biotinylated conjugate that localizes
to the sites of antigen
Avidin which is chemically conjugated to
horseradish peroxidase, is added;
avidin binds tightly to the biotinylated antibody
localizing the peroxidase moiety at the site of
antigen in the tissue section.

25. Advantage: Rapid
Disadvantage:
◦ 1) Different batches of biotin and different batches of avidin have
◦ differing affinities for one other → affects the sensitivity
◦ 2) Produces non- specific (false-positive) staining.

26. (STREPT) AVIDIN-BIOTIN TECHNIQUES
◦ The labeled streptavidin-biotin technique is the most widely used methodology in diagnostic
immunohistochemistry.
◦ 3 -step technique:
1. unconjugated primary antibody as the first layer,
2. followed by a biotinylated secondary antibody.
3. The third layer is either a complex of enzyme-labeled biotin and streptavidin, or enzyme-
labeled streptavidin
◦ The enzyme can be either horseradish peroxidase or alkaline phosphatase, used with a
chromogen of choice
◦ Streptavidin has now largely replaced the use of avidin in immunohistochemical detection
techniques.

27. Streptomyces
avidini

28. HAPTEN LABELING TECHNIQUE
BIOTINYLATED TYRAMIDE SIGNAL AMPLIFICATION
◦ Bobrow et al. first described the use of biotinylated tyramide to enhance signal
amplification, in 1989.
◦ The technique is based around the streptavidin biotin technique.
◦ Advantage: Enables many antigens which had previously been unreactive in formalin-
fixed paraffin-embedded tissue to be demonstrated.
◦ Disadvantage: Excessive background staining

29. PROCEDURE Application of the primary antibody
subsequent incubations in biotinylated
secondary antibody
horseradish peroxidase-labeled streptavidin
subsequent treatment with the biotinylated
tyramide amplification reagent
free biotin radicals

30. BIOTIN FREE CATALYSED SIGNAL AMPLIFICATION (CSA II)
• Introduced by DAKO to reduce the problems associated with endogenous biotin in
conventional tyramide signal amplification system
• Following incubation in primary antibody, a secondary anti mouse immunoglobulin
conjugated with horse radish peroxidase is bound to the primary antibody
• The third layer involves peroxidase- catalyzed deposition of fluorescyl tyramide, which in
turn is reacted with peroxidase conjugated antifluorescin, producing a greatly enhanced
signal

31. Tissue fixation
A prerequisite for all routine histological and cytological investigations is to ensure
preservation of tissue architecture and cell morphology by adequate and appropriate
fixation.
• The fixative should preserve antigenic integrity and should limit extraction, diffusion,
or displacement of antigen during subsequent processing.
• Fixation prevents the autolysis and necrosis of excised tissues, enhances the
refractive index of tissue constituents and increases the resistance of cellular elements to
tissue processing.
◦ Show good preservation of morphologic details after embedding in a support medium
(e.g., paraffin).

32. • Good fixation is the delicate balance between under-fixation and overfixation.
• Ideal fixation is the balance between good morphology and good antigenicity.
• Prompt fixation is essential to achieve consistent results.
• Poor fixation or delay in fixation - loss of antigenicity or diffusion of antigens into the
surrounding tissue.
◦ Fixative - changes in the steric configuration of proteins, which may mask antigenic sites
(epitopes) and adversely affect binding with antibody.
◦ Cross-linking fixatives (formaldehyde) alter the IHC results for a significant number of
antigens, whereas coagulant fixatives (ethanol) have been reported to produce fewer
changes, although there remains some controversy.
A robust and optimized fixation protocol is a critical step in an immunohistochemistry protocol as
an antigen that has been inappropriately fixed may not be detected in downstream detection.

33. ◦ When formalin-based fixatives are used, intermolecular and intramolecular cross
linkages are formed with certain structural proteins. These are responsible for the
masking of the tissue antigens.
◦ Methylene bridges forms between reactive sites on tissue proteins.
The most popular choice of fixatives for routine histology are formalin based, either as a 10%
solution or with the addition of different chemical constituents

34. UNMASKING OF ANTIGEN
SITES

35. MANUAL METHODS FOR ANTIGEN UNMASKING
Proteolytic enzyme digestion
Microwave oven irradiation
Combined microwave oven
irradiation and proteolytic enzyme
digestion
Pressure cooker heating
Decloaker heating
Pressure cooker inside a
microwave oven
Autoclave heating
Water bath heating
Steamer heating
Before antigen
unmasking
pretreatments are
employed, the
sections are
dewaxed, rinsed
in alcohol, and
washed in water.

36. PROTEOLYTIC ENZYME DIGESTION
• Described by Huang et al. (1976), Curran and Gregory (1977), and Mepham et al. (1979).
• Principle- Digestion breaks down formalin cross-linking and hence the antigenic sites for
a number of antibodies are uncovered.
• Under-digestion results in too little staining, because the antigens are not fully exposed.
• Over-digestion can produce false positive staining, high background levels, and tissue
damage.
The most popular enzymes employed today are trypsin and protease, but other proteolytic
enzymes such as chymotrypsin, pronase, proteinase K, and pepsin may also be used

37. HEAT-MEDIATED ANTIGEN RETRIEVAL TECHNIQUES
o great improvement in the quality and reproducibility of immunohistochemistry.
◦ widened its use as an important diagnostic tool in histopathology
During formalin fixation intermolecular and methylene bridges and weak Schiff bases form
intramolecular cross-linkages, which may prevent it from being recognized by a specific antibody. Heat-
mediated antigen retrieval removes the weaker Schiff bases but does not affect the methylene bridges, so
the resulting protein conformation is intermediate between fixed and unfixed.
Morgan et al. (1997), who postulated that calcium coordination complexes formed during formalin
fixation prevent antibodies from combining with epitopes on tissue-bound antigens. High temperature
weakens or breaks some of the calcium coordinate bonds, but the effect is reversible on cooling
Heavy metal salts (as described by Shi et al. 1991) act as a protein precipitant, forming insoluble
complexes with polypeptides, and that protein precipitating fixatives display better preservation of
antigens than do cross-linking aldehyde fixatives
1
2
3

38. MICROWAVE ANTIGEN RETRIEVAL
• Shi et al. (1991) first established the use of microwave heating for antigen retrieval.
• Gerdes et al. (1992) used microwave antigen retrieval with a non-toxic citrate buffer at pH 6.0 .
• Cattoretti et al. (1993) established microwave oven heating as an alternative to proteolytic enzyme
digestion.
• Antigen retrieval solutions: 0.01 M citrate buffer at pH 6.0 and 0.1 mMEDTA at pH 8.0
• Uneven heating and the production of hot spots

39. PRESSURE COOKER ANTIGEN RETRIEVAL
• Norton et al. (1994) suggested the use of the pressure cooker as an alternative to the
microwave oven. Batch variation and production of hot and cold spots in the microwave oven
could be overcome.
• Pressure cooking is said to be more uniform than other heating methods.
• A pressure cooker at 15 psi (10.3 kPa) reaches a temperature
of around 120°C at full pressure
• It is preferable to use a stainless steel domestic
pressure cooker, because aluminum pressure
cookers are susceptibleto corrosion from some
of the antigen retrieval buffers

40. Steamer
• Steam heating appears to be less efficient than either microwave oven heating or pressure
cooking
• Advantage - less damaging to tissues than the other heating methods
Autoclave
• This method offers an alternative form of heat mediated antigen retrieval, producing good
results for nuclear antigens such as MIB1, p21 and p53

41. Water bath
• Kawai et al. (1994) demonstrated that a water bath set at 90°C was adequate for antigen
retrieval.
• Increasing the temperature to 95–98°C, antigen retrieval was improved and the
incubation times could be decreased.
Advantage –
• gentler on the tissue sections because the temperature
is set below boiling point.
• antigen retrieval buffer does not evaporate and
• expensive commercial antigen retrieval solutions
can be safely reused
Disadvantage -antigen retrieval time is increased

42. COMBINED MICROWAVE ANTIGEN RETRIEVAL AND TRYPSIN
DIGESTION
• Infrequently used today;
• Brief proteolytic digestion can be carried out before or after microwave irradiation.
Advantages of heat pretreatment
• Some antigens previously thought lost in routinely processed
paraffin embedded sections are now recovered by heat pretreatment.
• Many antigens are retrieved by uniform heating times, regardless of length of fixation.

43. PITFALLS OF HEAT PRETREATMENT
• Care should be taken not to allow the sections to dry after heating, as this destroys antigenicity.
• The boiling of poorly fixed material also damages nuclear detail.
• Fibrous and fatty tissues tend to detach from the slide.
Prevention:
• Vectabond or APES-coated slides (3-Aminopropylenetriethoxysilane) can be dipped in 10% formal
saline for 1–2 minutes and air dried before picking up the sections.

44. COMMERCIALANTIGEN RETRIEVAL SOLUTIONS
•. They can be either specialized high pH solutions (recommended for certain antibodies) or lower pH 6.0 for more general use.
Citrate buffer pH 6.0
EDTA buffer pH 8.5
Pepsin reagent
Tris-HCL buffer pH- 10

45. DETECTION OF LOW LEVELS OF ANTIGEN
ENHANCEMENT AND AMPLIFICATION
oOptimum dilution will depend on type
and duration of fixation
concentration of primary antibody =
optimum dilution of primary antibody

46. IMMUNOHISTOCHEMISTRY IN PRACTICE
• The choice of technique to suit the needs of particular types of work is governed by some important
factors.
Frozen sections
• Although the use of frozen sections for diagnostic purposes is decreasing, immunohistochemistry on
frozen sections remains an important histological tool.
Advantage: preserves enzyme and antigen function
Disadvantages:
• Poor morphology
• Limited prospective studies
• Storage of material difficult
• Cutting difficulty over paraffin sections

47. • Poor morphology → improved by ensuring the frozen sections are thoroughly dried both before and
after the sections are fixed in acetone.
• Acetone assists preservation of the antigen and related morphology and also destroys most harmful
infective agents.

48. CYTOLOGICAL PREPARATIONS
• Acetone-fixed smears are often preferred by the immunohistochemist as it allows a wide range of
primary antibodies to be employed without destroying the target epitopes
• In alcohol, consequently the number of antigens demonstrable may be limited, although perhaps
the morphology is superior.

49. BLOCKING ENDOGENOUS ENZYMES
• If enzymes similar to those used as the antibody label are present in the tissue, they may react with
the substrate used to localize the tracer and give rise to problems in interpretation.
• Inhibiting endogenous enzyme activity prior to staining can eliminate false-positive reactions.
• Tissues incubated with DAB substrate prior to primary antibody incubation- if tissue turns brown-
peroxidase present and blocking steps needed.
• Incubation in absolute methanol containing 0.5% hydrogen peroxide for 10 minutes at room
temperature.
Endogenous alkaline phosphatase activity can be blocked by - Adding levamisole in the final
incubating medium. Using 20%acetic acid can block intestinal alkaline phosphatase.
Proteins blocked by: 10%normal serum

50. Blocking background staining
• The major causes of background staining in immunohistochemistry are
• Non-specific uptake of antigen, particularly the high affinity of collagen and reticulin for
immunoglobulins, can cause high levels of background staining
hydrophobic and ionic
interactions
endogenous enzyme
activity

51. Hydrophobic interactions :
Non-specific staining is most commonly produced because the primary antibody is attracted
nonimmunologically to highly charged groups present on connective tissue elements.
• Prevention: Add an innocuous protein solution to the section before applying the primary antibody.
• Traditionally, non-immune serum from the animal species in which the second (bridging) antibody
was raised is used as a blocking serum
• collagen
• connective
tissues
• epithelium
• adipocytes.
Addition of a blocking protein,
• Addition of a detergent such as Triton X ,
• Addition of a high salt concentration, 2.5%NaCl, to the
buffer.
• Addition of the blocking serum to the diluted primary
antibody
PREVENTION

52. CONTROLS:
• Controls validate immunohistochemical results.
• It is essential that any method using immunohistochemistry principles include controls to test for
the specificity of the antibodies involved.
Negative control. This involves either the omission of the primary antibody from the staining
schedule or the replacement of the specific primary antibody by an immunoglobulin which is
directed against an unrelated antigen
Positive control. Cells or tissues that are known to contain the specific Ag detects false
negatives due to fixation and processing. It is used to validate the protocol or procedure used

53. COUNTERSTAINING
◦ The final step in the process is counterstaining and mounting slides.
◦ Counterstains used are: Haematoxylin , Hoechst stain and DAPI (4',6-diamidino-2-phenylindole)
◦ Hematoxylin is used as the nuclear counterstain for most routine IHC staining
◦ Hoechst stains - family of blue fluorescent
dyes used to stain DNA. There are three related
Hoechst stains: Hoechst 33258, Hoechst 33342,
and Hoechst 34580.
• DAPI (4',6-diamidino-2-phenylindole) is a
fluorescent stain. It is used extensively in
fluorescence microscopy. As DAPI can pass
through an intact cell membrane, it can be
used to stain both live and fixed cells.

54. IMMUNOHISTOCHEMISTRY
QUALITY CONTROL

55. FACTORS AFFECTING STAIN QUALITY
TISSUE FACTORS
FIXATION-
• the purpose of fixation is to preserve tissue and prevent further degradation by the
action of enzymes or microorganisms
• Prolonged fixation- irretrievable loss of many antigens such as CD 20 and
immunoglobulin Ig
• Delayed fixation- detrimental to antigens
• Formalin- most universal of fixatives
10% formal saline, 10% NBF ( except for CD45RO) and 10% zinc formalin (except for CD3)
Alcohol based fixatives

56. PROCESSING
• Inadequately processed- poor quality sections, with poor adhesion to slides (fatty tissues)
• High temperature- detrimental to antigens that are heat labile
• Microwave processing is used to speed processing time and reduce turnaround time
REAGENT FACTORS
• Correct storage, handling and application of the reagents used
Paraffin with a low temperature melting point

57. BUFFERS AND DILUENTS
• pH should be monitored
• Many antibody dilents contain additives such as sodium azide to stabilize and maintain
protein
• Additives may inhibit or interfere with staining if present at excess levels
ANTIBODIES
◦ Storage temperature of antibody is critical
◦ Concentrated antibodies have longer shelf life
◦ Concentrated antibodies can be mixed with glycerine to prevent ice crystal formation,
aliquoted into cryovials , then snap frozen and stored at -80̊Cfreezer – extends shelf life
◦ Frost freeze -20̊C freezers should be avoided

58. PROCEDURAL FACTORS
BLOCK AND SLIDE STORAGE CONDITIONS
◦ Processed blocks- cool, dry places
◦ Resealing paraffin blocks after cutting – prevents physical damage
◦ The viability of the antigen and speed of antigen deterioration in cut tissue sections is
highly dependent upon the antigen under consideration and the temperature used for
section adhesion
◦ It is advisable to dry slides at 37̊C
◦ Urgent cases- 60̊C for upto 4 hours
◦ HER2- sections should not be dried at 60̊C more than 1 hours

59. MONITORING STAIN QUALITY
VALIDATION OF ANTIBODIES
• Most commercial antibodies have data sheets available on-line which should include a number
of facts for consideration
• These should indicate the host in which the antibody was raised (eg.rabbit, mouse or goat),
location of the target antigen, concentration of the antibody, recommended application (eg.
frozen tissue , formalin fixed paraffin embedded tissue), recommended positive and negative
control tissue sources, classification (eg. Analyte-specific reagent, research use only, or invitro
diagnostics)
• Selection of the most appropriate epitope retrieval method is important to ensure maximum
sensitivity of the stain
• Antibodies are pH sensitive and has been shown that staining intensity is better when the epi

60. • Staining protocol generally consists of trial and error
• If the stain is too strong then further dilution of the primary antibody may improve
staining specificity
• If target antigen staining fades and the background remains, then the addition of a
blocking step, change of epitope retrieval or changing the detection system used may
improve the result
• Changing the antibody diluent may help to increase the antibody’s reactivity, while at the
same time lowering background staining
• Weak staining may be improved with an increase of the antibody concentration
• Negative staining- change one variable at a time and document each reagent, step and
reaction time

61. • Negative staining may be resolved by increasing the antibody concentration, changing the
epitope retrieval method, changing the solution pH, changing the epitope retrieval method,
changing the antibody diluent to one of a different pH, changing the base composition or
using an amplification step as a part of the detection system.
• Over fixation- require more aggressive epitope retrieval method
• False positive staining of negative tissue controls suggests that the concentration of the
antibody is too high

62. CONTROL SLIDES
oControl slides include both reagent substitution and internal or external tissue controls
oControl tissue selection should be supported by publications and the selection process to be
documented
INTERNAL AND EXTERNAL POSITIVE CONTROLS
oMany tissues contain native components that serve as internal positive controls for
immunohistochemical staining. Eg.crypts of normal colon stain with polyclonal CEA
oInternal controls are better than external controls
DAILY SLIDE REVIEW
oAll slides should be reviewed and quality assessed prior to being sent out of the laboratory
EXTERNAL QUALITY ASSURANCE
oInter laboratory consistency of quality and results should be assessed

63. TROUBLE SHOOTING
FALSE NEGATIVE STAINING
• Process failure
• Positive control selection
• Incomplete deparaffinization
• Epitope retrieval
• Temperature
• Antibody preparation
• Chromogen incompatibility
FALSE POSITIVE STAINING
• Poor quality of fixation
• Technical preparation
• Tissue drying
• Intrinsic tissue factors
• Antibody concentration
• Detection system
• Chromogen
• Species cross reactivity
• Automation error

64. FALSE POSITIVE STAINING FALSE NEGATIVE STAINING

65. LIMITATIONS OF IMMUNOHISTOCHEMISTRY:
1. Experience:
2. Availability of antibodies: The paucity of antibody with high degree of
specificity for cellular and tissue antigens
3. Antigen loss: The specificity of an antibody for particular antigen and its
ability to react with that
antigen require the preservation of antigen configuration

66. CURRENT APPLICATIONS OF IMMUNOHISTOCHEMISTRY
Tumor Pathology
• Classification of Neoplasms
• Diagnosis of Malignancy
• Prognostic Markers
• Predicting response to
treatment
• Detection of metastases
• Screening of inherited cancer
syndromes
Non- Tumor Pathology
• Neurodegenerative diseases
• Brain trauma
• Muscle diseases
• Amyloidosis
• Dementias

67. REFERENCES
1. Kim SW, Roh J, Park CS. Immunohistochemistry for pathologists: protocols,
pitfalls, and tips. Journal of pathology and translational medicine. 2016
Nov;50(6):411.
2. Suvarna KS, Layton C, Bancroft JD, editors. Bancroft's theory and practice of
histological techniques E-Book. Elsevier Health Sciences; 2018 Feb 27.
3. Culling CF. Handbook of histopathological and histochemical techniques: including
museum techniques. Butterworth-Heinemann; 2013 Oct 22.