the basics of IHC

1. ‫دهؤك‬‫يا‬ ‫ثوليتةكنيكى‬ ‫انكويا‬‫ز‬
‫ـنية‬‫ـ‬‫ق‬‫الت‬ ‫ـوك‬‫ـ‬‫ه‬‫د‬ ‫ـة‬‫ع‬‫جام‬
Duhok Polytechnic University
Lab 8 : Immunohistochemistry (II)
4th year students
Practical Immunopathology
Ass. lecturer: Ahmed Jumaa Ahmed
M. Sc. Immunology and Allergy 15 /04/2018
Duhok Polytechnic University
Shekhan Technical College of Health
Dept. of Medical Laboratory Technology

2. Ag Retrieval
Group Thinking
What does Ag Retrieval Mean?

3. Ag Retrieval
• These changes can often be overcome by antigen
retrieval methods.
• The section must be treated to expose buried
antigenic epitopes with either proteases or by
heating in
• However, the effects of fixation, including protein-protein and
protein-nucleic acid cross linking and calcium ion bonding, cause
damage epitopes through alteration of the protein structure (3D).

4. Ag Retrieval
• Function
1. To revers formaldehyde induced change of Ag.
2. To re-expose the Ag to the surface.
3. Increases the accessibility of the Ab to the Ag.
• Two methods:
1. Heat:-
• Heat Induced Epitope Retrieval (HIER) is widely used
• (60C)
2. Enzyme digestion:-
• Proteolytic-Induced Epitope Retrieval (PIER)
• Eg Proteinase K, Trypsin, Pepsin
Destroys some epitopes
May damage the morphology of the section.

5. Antibodies Incubation
1. Primary antibody
• Used in a solution at different dilutions with the
blocking agent.
• The incubation time changes according to
1. the properties of the antigen or antibody.
2. depending on the temperature.

6. Antibodies Incubation
2. Secondary antibody:
• Must be raised in the species not from:-
1. the species of which the cells or tissues are taken.
2. the species were the primary antibody is raised.
• It should specifically recognize the immunoglobuline
of the species in which the primary antibody is
raised.

7. Primary Antibodies
• Two types of antibodies are used in IHC
1. Monoclonal Abs
2. Polyclonal Abs

8. MONOCLONAL POLYCLONAL
1. Produced by Hybridization. 1. Produced by injecting an
animal with antigen and
harvesting the sera.
2. Contains one type of Ab which
is very pure and specific to one
epitope of an antigen.
2. Contains a mixture of
antibodies with high binding
affinity to different epitopes.
3. commonly raised in mice
(hybridoma)
3. Many different species (rabbit,
guinea pig, goat or sheep BUT
mostly rabbits)
4. Highly specific. 4. Less specific than monoclonal
Ab.

9. Antibody application or
Immunolabelling methods or
Antibody labelling methods:
• Incubation with primary and secondary antibody
– On slides on a stage in humid chamber

10. 1. Direct immunofluorescence Method
• One step
• The primary antibody is conjugated directly to the label.
• Ab is labeled with fluorescent tag (more commonly) or an enzyme.
• The labeled antibody reacts directly with the antigen in the
histological or cytological preparation.

11. Direct Method
Tissue Antigen
Labeled Antibody

12. 2. Indirect immunofluorescence
• Two steps
• Step 1 – bind 1° Ab to Ag
• Step 2 – bind fluorescent labeled 2° Ab to 1° Ab
• Primary antibody is unlabeled. A secondary antibody is labeled.
• Secondary antibody must be raised against the immunoglobulin of
the species which the primary antibody is made in.

13. Two-Step Indirect Method
Tissue Antigen
Primary Antibody
Secondary Antibody

14. Group Thinking
• What is the main difference between both methods
Or
• What is the importance of the 2° Ab

15. 3. Indirect immunoenzyme
(Peroxidase anti-peroxidase)(PAP) method
• Three steps
• Step 1 – bind 1° Ab to Ag
• Step 2 – bind unconjugated 2° Ab to 1° Ab
• Step 3 – bind PAP complex to 2° Ab
• Sensitive – signal amplification
• Use DAB as a substrate for peroxidase 
colourimetric end product

16. PAP Method
(peroxidase anti-peroxidase method)

17. 4. Indirect immunoenzyme
(Avidin-Biotin-Complex)(ABC) method
• Three steps
• Step 1 – bind 1° Ab to Ag
• Step 2 – bind biotinylated 2° Ab to 1° Ab
(The secondary antibody is labelled with biotin)
• Step 3 – bind avidin-biotin-peroxidase complex to
2° Ab
– 2° Ab binds to avidin in the avidin-biotin-enzym complex.

18. 4. Indirect immunoenzyme
(Avidin-Biotin-Complex)(ABC) method
• Very high sensitivity method - signal
amplification
• Used in research more than routine
studies.
• It is longer and more expensive.
• Use DAB as a chromogen substrate for
peroxidase  colourimetric end product
http://www.biologie.uni-
regensburg.de/Zoologie/Schneuwly/Hofb
auer/DROSI/strentw42.htm

20. Detection Methods or
Labelling Antibodies
• Antibodies are not visible with standard microscopy and
must be labeled in a manner that does not interfere with
their binding specificity.
Common labels include:-
1. Immunofluorescence method:
– A fluorochrome is used as the label.
– Fluorescein, Rodomine, FITC, AMCA, TRITC, Texas Red.
– For visualization:-
1) Fluorescence microscope with appropriate filter or
2) Confocal laser scanning microscope.

21. Detection Methods or Labelling
2. Immunoenzyme method:
– An enzyme is used as the label.
– Peroxidase, alkaline phosphatase, glucose oxidase
– Chromogen (staining chemical) must be used
• Enzyme labels produce a colored precipitate in the presence of a
specific substrate
• Diamino Benzidine (DAB) produces a dark brown precipitate.
• Alkaline phosphatase produces either red or blue precipitates.

22. Chromogens
• In order to visualize the enzymes labelling the antibodies with light
microscope, enzyme – substrate reactions, which convert colorless
chromogens into visible colored end-products, is used.
• The color of the reaction is determined by the selection of a
precipitating chromogen, with which the enzyme reacts
1. Peroxidase- hydrogen peroxide- diaminobenzidine (DAB) (dark
brown) (potential carcinogen)
2. aminoethylcarbazole (AEC) (red)
3. Chloro-naphthol (CN) (blue)
4. Hanker-Yates reagent( dark blue)
5. naphthol pyronin (red-purple)

23. • How we will know if the staining is successful
and all the background tissue is still out
there?

24. Counter Staining
• Provides contrast to the primary stain.
• Stain background , tissue,
• Most commonly used counter stain is Haematoxylin and Eosin
staining.
• Haematoxylin stains nucleic acids blue.
• Eosin stains eisonophilic structures in red or pink.

28. Neurobiotin Calbindin Merged image
50µm
Intrinsic primary afferent neurons of
the myenteric plexus are Dogiel Type
II neurons that express calbindin

29. IL-1b activates neurons and glia in
the submucosal plexus of the ileum.

30. Red (CY3): rat anti-substance P
Green (FITC): mouse anti-VIP
Blue (AMCA): rabbit anti-CGRP
Distribution of nerves in the guinea pig ileum submucosal plexus
TRIPLE LABELLING

32. Automation
• Fully automated IHC work stations are a common
• Advantages:
1. Greater consistency of staining
2. Fast and accurate results
3. Decreased use of reagents
4. Less use of man power

33. • Difference between IHC and Haematoxylin-eosin staining
H & E
IHC
1. Non specific reactions
1. highly specific for Ag
2. Based on chemical reactions
(Acid and Alkaline)
2. Based on Ag-Ab reaction
(Immunologic reactions)
3. Ordinary microscope is
needed
3. Special Microscope (UV) is
needed for observation.

34. http://www.immunohistochemistry.us/

35. THE END
ANY QUESTIONS