Immunohistochemistry is a technique that combines immunology and histology to detect antigens in tissues using the antibody-antigen interaction. It allows determination of antigen expression and location within tissues at the cellular level. The process involves using labeled antibodies that bind specifically to antigens in tissue sections or cell samples. This binding is then visualized through enzymatic reactions or use of fluorescent dyes, colloidal gold, or radioactive labels. Immunohistochemistry is widely used in research and clinical diagnosis and has various applications including disease diagnosis, prognosis, and therapeutic monitoring.

1. Immunohistochemistry

2. Immunohistochemistry
• Introduction;
• Immunohistochemistry is as the name implies, a combination of two
disciplines – immunology and histology. The Immunohistochemistry
technique is used not only to determine if a tissue express or does not
express a particular antigen but also to determine the antigenic status of
particular cells within that tissue and microantomic location of the antigen.
• Immunohistochemistry uses antibodies to distinguish antigenic difference
between the cells.
Definition ;
• This is a technique for identifying cellular or tissue constituents (antigens)
by means of antigen antibody interactions, the site of antibody binding
being identified either by direct labeling of the antibody, or by use of a
secondary labeling method.Im
• munohistochemistry- using tissue sections.
• Immunocytochemistry - cytological preparations

3. Continuous..
Antigen ;
In immunology, an antigen is a molecule or molecular structure or any foreign
particulate matter or a pollen grain that can bind to a specific antibody or T-
cell receptor. The presence of antigens in the body may trigger an immune
response.
Antibody ;
An antibody, also known as an immunoglobulin, is a large, Y-shaped protein
used by the immune system to identify and neutralize foreign objects such as
pathogenic bacteria and viruses. The antibody recognizes a unique molecule
of the pathogen, called an antigen.

4. Continuous
Affinity;
Affinity is the three-dimensional fit of the antibody to its specific antigen and
is a measure of the binding strength between the antigenic epitope and its
specific antibody-combining site.
Avidity;
Avidity is a related property referring to the heterogeneity of the antiserum
which will contain various antibodies reacting with different epitopes of the
antigen molecule. Avidity therefore is the functional combining strength of an
antibody with its antigen.

5. Principle of immunohistochemistry
Principle of immunohistochemistry;
• Immunohistochemistry is a method for localizing specific antigen in tissues
or cells based on antigen antibody reaction. The site of antibody binding is
identified either by tagging the antibody, directly or indirectly with a visible
label.
• Fluorescent dye, colloidal metal, hapten, radioactive marker.

6. IHC sample preparation
Sample preparation:
IHC can be broadly classified into two forms based on the type of tissue
processing involved: IHC- formalin-fixed,paraffin-embedded (FFPE) and IHC-
frozen (Fr). Often the preservation method is closely associated with the type of
fixation. Formalin-fixed tissues are commonly paraffin-embedded following
fixation, while frozen tissue sections can be fixed with formaldehyde or alcohol
prior to or following cryosectioning.

7. IHC sample preparation
Flowchart;

8. Fixatives used in immunohistochemistry;
Standard fixatives;
Aldehydes;
4% formaldehyde in phosphate-buffered saline (PBS) is the most common
fixative for preserving protein targets in tissues. Formaldehyde reacts with
amino groups in proteins to form methylene bridges that crosslink proteins in
tissue sections. These molecular crosslinks can mask protein epitopes from
antibody binding and may require the addition of an antigen retrieval step for
proper IHC staining. Additionally, formaldehyde-mediated tissue fixation has
been shown to induce translocation of phosphorylation-dependent epitopes
from the membrane to the cytoplasm.
Alcohols;
The predominant alcohols used for fixation are ≥70% methanol and ≥80%
ethanol. Alcohols work by removing and replacing water molecules in tissue,
which can destabilize Hydrophobic bonds and alter the tertiary structure of
proteins. This also causes the Precipitation of soluble proteins, making
alcohol-mediated fixation more appropriate for detection of membrane bound
proteins.

9. Fixatives used in immunohistochemistry
Acetone;
Acetone is also used as a strong dehydrant and precipitant, typically applied
to sections of snap-frozen tissues. Acetone fixation is generally mild and may
be followed by fixation with alcohols or formaldehyde.
What Is difference between paraformaldehyde , Formaldehyde and
formalin?
Paraformaldehyde (PFA) is the polymerized form of formaldehyde and is not
itself a fixing agent. Formaldehyde can be prepared by dissolving PFA in PBS
using heat and sodium hydroxide (NaOH). Formalin refers to a saturated
formaldehyde solution and some commercial formalin solutions include
methanol as a stabilizer to prevent formaldehyde polymerization. A 10%
formalin solution is equivalent to a 3.7% formaldehyde solution

10. Labels used in immunohistochemistry
Enzyme labels;
Enzymes are the most widely used labels in immunohistochemistry, and
incubation with a chromogen using a standard histochemical method
produces a stable, colored reaction end-product suitable for the light
microscope
Horseradish peroxidase is the most widely used enzyme, and in
combination with the most favored chromogen, i.e. 3,30diaminobenzidene
tetrahydrochloride (DAB).
Horseradish peroxidase is commonly used as an antibody label for several
reasons;
• its small size does not hinder the binding of antibodies to adjacent sites.
• Chance of contamination is minimized.
• Stable enzyme.Endogenous activity is easily quenched.
• Calf intestinal alkaline phosphatase is the most widely used alternative
enzyme tracer to horseradish peroxidase,particularlysince the development
of the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method in
1984 by Cordell.

11. Labels used in immunohistochemistry
• Bacterial-derived B-D-galactosidase has also been used as a
tracerColloidal metal labelsWhen used alone, colloidal gold conjugates
appear pink when viewed using the light microscope. An silver
precipitation reaction can be used to amplify the visibility of the gold
conjugates.
• Fluorescent labels
• Radiolabels
Chromogens;
3,3a-diaminobenzidene tetrahydrochloride (DAB), it yields a crisp, insoluble,
stable, dark brown reaction end-product. Although DAB has been reported to
be a potential carcinogen, the risk is now thought to be low.
• 3-amino-9-ethylcarbazole –red
• 4-chloro-1-naphthol - blue;
• Hanker-Yates reagent
• dark blue? a-naphthol pyronin - red-purple

12. Buffer mediums;

13. Fixation technique in immunohistochemistry

14. Antibodies detection methods:
Monoclonal antibody;
A monoclonal antibody is an antibody made by cloning a unique white blood
cell. All subsequent antibodies derived this way trace back to a unique parent
cell. Monoclonal antibodies can have monovalent affinity, binding only to the
same epitope.
Polyclonal Antibody;
Polyclonal antibodies (pAbs) are a complex mixture of several antibodies that
are usually produced by different B-cell clones of an animal. These antibodies
recognize and bind to many different epitopes of a single antigen and hence
can form lattices with the antigens.
Antibody detection :
Frequently, IHC uses the indirect method of detection in which a secondary
antibody, directed against the constant region of primary antibody, carries the
label. The indirect method is more sensitive than using a directly labeled
primary antibody because multiple labeled secondary antibodies can bind to a
single primary antibody. After incubating the tissue sample with the appropriate
labeled antibody, the antigen can be detected by IF or by a chromogenic
reaction

15. Immunohistochemical methods ;
• 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.
New direct technique;
• New Direct technique Pluzek et al in 1993
• . Commercial name- Enhanced Polymer One-step Staining (EPOS)
• . A large number of primary antibody molecules and peroxidase enzymes
are attached to a dextran polymer ‘backbone’, hence increasing the
signal amplification and provide greater sensitivity.

16. Continuous,,,
Two step indirect technique;
A labeled secondary antibody directed against the immunoglobulinof 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.
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.

17. Unlabelled antibody methods;
Peroxidase anti peroxidase 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.
Alkaline phosphatase Anti alkaline phosphatase method /APAAP
Principle same as those described for the PAP method except that the PAP
complex is replaced with an APAAP complex.
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

18. Biotin/,Avidin procedure;
• A biotinylated enzyme (HRP or AP) is pre-incubated with free avidin to form
large avidin–biotin–enzyme complexes. Typically, the avidin–biotinylated
enzyme are mixed together in a specified ratio to prevent avidin saturation
and incubated for about 15 minutes at room temperature to form the
complex.

19. Epitope /Antigen retrieval methods ;
• Masked epitopes can be recovered with an antigen retrieval step, which
works to promote epitope availability and enhance
• immunogenicity (Figure 2). Proteolytic-Induced Epitope Retrieval (PIER)
and Heat-Induced Epitope Retrieval (HIER) are two
• of the most widely used antigen retrieval methods for FFPE tissue
sections.

20. Epitope retrieval protocols;

21. Epitope retrieval protocols;
Different antigen retrieval methods
1 . Microwave oven using EDTA (PH 8)or citrate buffer( PH 6.5)
2. Pressure cooker for gentle tissue at 10.3 kpa
3. Steamers
4.Water bath at 95 -98 degree Celsius is most optimum for antigen retrieval

22. 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.
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.

23. Immunohistochemistry in practice;
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.

24. Limitations of immunohistochemistry
Experience; Experience is critical in standardizing the procedure including
the selection and proper dilutions of necessary reagents and regular
performance of all the appropriate controls. Interpretation too has its
foundation in experience.
Availibility of antibodies;The paucity of antibody with high degree of
specificity for cellular and tissue antigens was serious limitation until recently.
This has been remedied in part by using hybridoma technique for monoclonal
antibodies.
Antigen loss; The specificity of an antibody for particular antigen and its
ability to react with that antigen require the preservation of antigen
configuration

25. Applications of immunohistochemistry;