2. Definition
Principles Of Immunohistochemistry
Procedures Of Immunohistochemistry
Steps To Better Immunohistochemistry
Application Of Immunohistochemistry
Markers In Immunohistochemistry And
Application
Conclusion
References
2
3. Immunohistochemistry (IHC) is an important application of
monoclonal as well as polyclonal antibodies to determine the tissue
distribution of an antigen of interest in health and disease
The antibody-antigen interaction is visualized using either
chromogenic detection with a colored enzyme substrate such as
Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), or
fluorescent detection with a fluorescent dye (Duraiyan et al., 2012).
3
4. The principle of IHC has existed since the 1930s, but it was not
until 1941 that the first IHC study was reported. Coons and his
colleagues used Fluorescein isothiocyanate (FITC)-labeled
antibodies with a fluorescent dye to localize pneumococcal antigens
in infected tissues. Immunohistochemistry is carried out by
exploiting the principle that antibodies bind specifically to an
antigen in biological tissues, for example in the jaw, gonads or
heart (Duraiyan et al., 2012).
4
5. TISSUE PROCESSING, FIXATION, AND SECTIONING
Tissue fixation preserves antigens and prevents autolysis and
necrosis of harvested tissues.
Paraffin embedding is the most common method, but frozen and
floating sections are also available.
Section tissue at 4-7 μm thickness, and mount on adhesion-treated
slides (Abcam, 2022).
5
6. 6
Fig 1.0: Diagrammatic representation of the key steps involved in IHC (Verma et al., 2022).
7. ANTIGEN RETRIEVAL
Antigen retrieval methods include heat induced antigen retrieval
HIAR and enzymatic (protease-induced)
In a microwave oven, deparaffinized and rehydrated slides are
placed in 3% hydrogen peroxide for 5 minutes, then washed with
deionized water. The slides are then placed in retrieval buffer and
heated in the microwave at 100°C for 5-10 minutes. The slides are
then cooled and washed twice (Magaki et al., 2019)
The antigen retrieval reagent requires citrate buffer solution, PBS
buffer, EDTA, Tris-EDTA, and Tris-HCl (Abcam, 2021).
7
8. BLOCKING PROTEINS
Protein blocking step is required to reduce unwanted background
staining.
An ideal agent for the protein blocking is 5%–10% normal serum
from the same species of secondary antibody. Other agents include
protein buffers such as 0.1%–0.5% bovine serum albumin, gelatin,
or non-fat dry milk (Kim et al., 2016).
8
9. ANTIBODY SELECTION AND VALIDATION
Polyclonal and monoclonal antibodies are used for specific detection,
with polyclonal antibodies created by injecting a protein or peptide
fragment into animals, isolating antibodies from serum, and
recognizing multiple epitopes, respectively, monoclonal antibodies
react to a single epitope in an antigen (Kim et al., 2016).
9
10. TARGET ANTIGEN DETECTION METHODS
The direct method is a one-step staining method and involves a
labeled antibody reacting directly with the antigen in tissue sections.
The indirect method involves an unlabeled primary antibody (first
layer) that binds to the target antigen in the tissue and a
labeled secondary antibody (second layer) that reacts with the
primary antibody (Kim et al., 2016).
10
11. • Commonly used detection systems are as follows: avidinbiotin
complex method, labeled streptavidin biotin method, phosphatase
anti-phosphatase method, polymer-based detection system, and
tyramine amplification system (Kim et al., 2016).
11
12. 12
Fig 2.0: Illustration of polymeric amplification system. DAB, diaminobenzidine; HRP, horseradish peroxidase
(Kim et al., 2016).
13. ANTIBODY APPLICATION
Use serial dilutions of a concentrated antibody to determine the
optimal concentration of an antibody .
Apply 200μl of diluted primary antibody to slides and incubate at
room temperature for 80 minutes.
Wash in three changes of 0.1% TBS-Tween using different staining
dishes for each wash.
Apply species-specific secondary antibody and let sit for 15
minutes.
Wash with 0.1% TBS-Tween, add orange horseradish peroxidase,
and wash in three changes of 0.1% TBS-Tween for each wash
(Magaki et al., 2019).
13
15. COUNTERSTAINING, DEHYDRATION, CLEARING, AND
MOUNTING
Slides are placed in hematoxylin, rinsed in distilled water, and
incubated in Bluing solution.
After drying, they are immersed in three washes of Xylene, each
lasting 5 minutes.
The slides are then mounted in a permount and coverslip (Kim et
al., 2016).
15
19. Prognostic markers in cancers
Tumors of uncertain histogenesis
Prediction of response to therapy
Infections
In genetics
Neurodegenerative disorders
In muscle diseases
Research application (Duraiyan et al., 2012).
19
20. ◦ Kiel (ki-67) is a prognostic indicator for tumors
◦ Cytokeratin identifies epithelial cells
◦ Cluster of differentiation (CD20) detect B-cells, especially in
lymphomas
◦ Estrogen Receptors evaluate hormonal status in breast cancer
(Schurch et al., 2023)
20
21. ◦ Human epidermal growth factor receptor 2 (HER2) status for breast
and gastric cancers
◦ Glial fibrillary acidic protein (GFAP) for astrocytic tumors
◦ CD4 for T-helper cells
◦ CD8 for cytotoxic T-cells
◦ CD15 for granulocytes (Schurch et al., 2023).
21
22. IHC is a crucial tool for pathologists to understand disease
pathophysiology and validate biomarker discovery, leading to
personalized medicine. Despite recent automation and
standardization, optimization and interpretation are essential for
newly discovered molecules or antibodies. Validating specificity and
sensitivity is crucial, and a full literature review is recommended
before starting experiments. Careful planning and stabilizing inter
observer consistency are also essential for objectifying IHC results.
22
23. Buza, N. and Hui, P. (2017). Immunohistochemistry in Gynecologic Pathology: An Example-Based
Practical Update. Arch Pathol Lab Med,.141(8), 1052–1071.
Duraiyan, J., Govindarajan, R., Kaliyappan, K. and Palanisamy, M. (2012). Applications of
immunohistochemistry. Journal of Pharmacy & Bioallied Sciences. 4( 2), S307–S309.
Hawes, D., Shi, S.-R., Dabbs, D. J., Taylor, C. R. and Cote, R. J. (2009). Modern Surgical Pathology
(Second Edition). 1, 48–70.
Kim, S.-W., Roh, J. and Park, C.-S. (2016). Immunohistochemistry for Pathologists: Protocols, Pitfalls,
and Tips. Journal of Pathology and Translational Medicine. 50(6), 411–418.
Magaki, S., Hojat, S. A., Wei, B., So, A. and Yong, W. H. (2019). An Introduction to the Performance of
Immunohistochemistry. Methods Mol Biol. 1897, 289–298.
Masuda, S. and Nakanishi, Y. (2023). Application of Immunohistochemistry in Clinical Practices as a
Standardized Assay for Breast Cancer. Acta Histochem Cytochem. 56(1), 1–8.
Sheffield, B. S. (2016). Immunohistochemistry as a Practical Tool in Molecular Pathology. Arch Pathol
Lab Med. 140(8), 766–769.
23
