introduction to immunohistochemistry

1. Overview of immunohistochemistry& its
application
Overview of immunohistochemistry
&
its application
Ms.Sneha Durugkar
Reg no. PC/2017-X/170
M.S. Pharmacology & Toxicology
NIPER, Guwahati
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2. Contents
Introduction
History
Principle
Steps
Methods
Troubleshooting
Application
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3. Introduction
 Immunohistochemistry (IHC) staining is a laboratory technique that utilizes specific
antibodies for visualization of the quantity, distribution and cellular location of
immunogenic epitopes in histological tissue sections
Fig.1 IHC detection of cytokeratin 18 in human colon carcinoma
thermofisher.com/content/dam/LifeTech/Images/integration/000007-IHC-Cytokeratin-DyLight-633-streptavidin-463px.jpg
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4. A Synergy Of 3 Scientific Disciplines
Immuno Histo Chemistry
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5. History
 1942 coons, Creech, Jones,Berliner
Developed indirect immunofluorscence method in order to demonstrate an antigen
(pneumococcal antigen in tissue)
 1959 Singer
Developed a method for conjugating ab’s to a label(ferritin)
 1966 Graham & Karnovsky
Developed a method for tagging an enzyme to an Ab(hosrseradish peroxidase)
 1967 Nakane & Pierce
Developed a antigen retrieval method
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6. Principle
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Tissue section
Staining tissue

7. Steps
Counterstaining
Detection
Antigen retrival
Inactivation
Paraffin embedding
Tissue sectioning
Fixation
Tissue isolation
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8. Hofman FM, Taylor CR. Immunohistochemistry. Current protocols in immunology. 2002:21-4.7/18/2018 8
Dewaxing Inactivation
Antigen
retrieval
Add substrate

9. a. Tissue Isolation
 Sources : biopsy, surgery, animal model and autopsy.
 As antigens may denature, disappear and diffuse, autopsy specimen should be
fixated as soon as possible so as not to influence its label.
 Precaution
 Use sharp knife and scissors to avoid extrusion damage
 Cutter should be flat, small and thin (Normal size is 1.0 cm × 1.0 cm × 0.2
cm)
 Collect samples from live animals and fix samples immediately after
wash
 Make paraffin-embedded tissue or frozen tissue immediately after
sectioning or store the tissues in liquid nitrogen container or refrigerator
at -70℃
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10. b. Tissue Fixation
 For immunohistochemistry (IHC) to succeed, it is essential that the morphology of the
tissues and cells is retained and that the antigenic sites remain accessible to the detection
reagents being use.
 Fixation plays four critical roles in immunohistochemistry:
a) It preserves and stabilizes cell morphology and tissue architecture.
b) It inactivates proteolytic enzymes that could otherwise degrade the sample.
c) It strengthens samples so that they can withstand further processing and staining
d) It protects samples against microbial contamination and possible decomposition
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11. Cont…
Antigen Fixatives
Protein, peptides, enzymes 4% w/v paraformaldehyde
Delicate tissue Boulins solution
Amino acid 4% parafor. – 1% glutaraldehyde
Liver, spleen, bone marrow Zenkers solution
Nucleic acid carnoy’s sol.
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12. Methods of fixation
Immersion
• The immersion method maintains the tissue in fixing solution (at 4℃ if
needed) for a specified period which is determined by the antigen stability
and type of fixing solution used..
• Biopsy and surgical specimens as well as other non-irrigation tissues
commonly employ this fixation method.
Irrigation
• This method has the ability to fix tissues fully and quickly, suppressing
the interference of endogenous peroxidase. Therefore, it is a method of
choice in animal experiments.
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13. d. Paraffin Embedding
Dehydration
• Eg ethanol , acetone
Transperentizing
• Eg xylene, benzene ,
toluene
Immersion
• At 54-64℃.
Embedding
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14. c. Sectioning & Mounting
• The tissue is typically cut into thin sections (5-10 μm)
or smaller pieces (for whole mount studies) to facilitate
further study.
• before sectioning chilling is necessary.
• Paraffin 5 micron thick light microscopy
https://i.ytimg.com/vi/XMjGZHEG4cY/maxresdefault.jpg7/18/2018 14

15. d.Inactivation & blocking
 Inactivation  Blocking
 By using milk casine, bovine sera ,
buffers
HRP
inactivation
Incubate the paraffin
embedded section in
3% H2O2 for 10 min
Incubate the frozen section or
cell section in solution
composed of methanol and
3% H2O2 (v/v: 4:1) for 30
min
AP Inactivation
Incubate the
sample section in
0.1 mM
Levamisole
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16. e. De -paraffinization & Antigen retrieval
HIER PIER
Advantages Maintains epitope Commonly used for difficult to
retrieve epitopes
Buffer Glycine hcl ,citric acid,
tris EDTA
Neutral buffer solutions are used of
enzymes such as pepsin, proteinase K
or trypsin
Incubation
Time
10-20 minutes 5-30 minutes
pH pH 8-10 pH 7.4
Precautions Heating can lead to
uneven antigen
retrieval
Heating can lead to uneven antigen
retrieval
Recommended
Antigens
No specific antigen Immunoglobulins, cytokeratins
Temperature 95°C 37°C
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17. 2.Detection
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Chromogenic
Fluorescent
Enzyme Substrate Colour
HRP DAB/AEC Brown
AP NBT/BCIP Red
eg. FITC
TRITC
AMCA

18. Methods
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Direct methodDirect method
Indirect method PAP method
ABC Method

19. Counterstaining
 To provide contrast to primary stain & can be cell structure specific.
 Added after Ab staining.
e.g. Haematoxylin
Eosin
Hoechst Fluorescent Stain
Nuclear Fast Red
Methyl Green
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20. Applications
IHC
Prognostic
Markers in
Cancer
Infectious &
Neurodegen-
erative
diseases
In GeneticsSurgical
pathology
Research
Application
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21. Troubleshooting
IHC
No staining
High
background
Non specific
staining
Poor or
damaged
Tissue
morphology
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22. Recent advances
Dual/multiple staining
Preparing direct abs
conjugates
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23. Summary
IHC is laboratory technique by which the it is easy to detect tissue structure ,
location and distribution of ag based on ag ab complex
By determining the presence or absence of specific marker within tumour
so gives clues of type and identification of neoplasm.
New advances in IHC makes it more sensitive, specific and useful for daily
laboratory uses.
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24. References
 Zieba A, Ponten F, Uhlén M, Landegren U. In situ protein detection with enhanced
specificity using DNA-conjugated antibodies and proximity ligation. Modern Pathology.
2017 Sep 22:modpathol 2017102
 Duraiyan J, Govindarajan R, Kaliyappan K, Palanisamy M. Applications of
immunohistochemistry. Journal of pharmacy & bioallied sciences. 2012 Aug;4(Suppl
2):S307.
 Bancroft JD, Gamble M, editors. Theory and practice of histological techniques. Elsevier
Health Sciences; 2008.
 Yeh IT, Mies C. Application of immunohistochemistry to breast lesions. Archives of
pathology & laboratory medicine. 2008 Mar;132(3):349-58.
 Haines DM, West KH. Immunohistochemistry: forging the links between immunology
and pathology. Veterinary immunology and immunopathology. 2005 Oct 18;108(1):151-6.
 Hofman FM, Taylor CR. Immunohistochemistry. Current protocols in immunology.
2002:21-4.
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