Immunohistochemistry, the basics and applications.pptx

the basics of IHC

1. زانكويا ثوليتةكنيكى يا دهؤك جامعـة دهــوك التقــنية Duhok Polytechnic University Lab 8 : Immunohistochemistry (II) 4 th year students Practical Immunopathology Ass. lecturer: Ahmed J umaa 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 To revers formaldehyde induced change of Ag. To re-expose the Ag to the surface. Increases the accessibility of the Ab to the Ag. Two methods: Heat :- Heat Induced Epitope Retrieval (HIER) is widely used (60C) 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 Primary antibody Used in a solution at different dilutions with the blocking agent. The incubation time changes according to the properties of the antigen or antibody. depending on the temperature.

6. Antibodies Incubation Secondary antibody: Must be raised in the species not from:- the species of which the cells or tissues are taken. 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 Monoclonal Abs Polyclonal Abs

8. MONOCLONAL POLYCLONAL Produced by Hybridization. Produced by injecting an animal with antigen and harvesting the sera. Contains one type of Ab which is very pure and specific to one epitope of an antigen. Contains a mixture of antibodies with high binding affinity to different epitopes. commonly raised in mice ( hybridoma ) Many different species ( rabbit, guinea pig, goat or sheep BUT mostly rabbits ) Highly specific. 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 immuno enzyme (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 immuno enzyme (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 immuno enzyme (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/Hofbauer/DROSI/strentw42.htm

19. 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:- Immuno fluorescence method: A fluorochrome is used as the label. Fluorescein, Rodomine , FITC, AMCA, TRITC, Texas Red. For visualization:- Fluorescence microscope with appropriate filter or Confocal laser scanning microscope .

20. Detection Methods or Labelling Immuno enzyme 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.

21. 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 Peroxidase- hydrogen peroxide- diaminobenzidine (DAB) ( dark brown ) (potential carcinogen) aminoethylcarbazole (AEC) ( red ) Chloro-naphthol (CN) ( blue ) Hanker-Yates reagent( dark blue ) naphthol pyronin ( red-purple )

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

23. 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.

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

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

26. 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

27. Automation Fully automated IHC work stations are a common Advantages : Greater consistency of staining Fast and accurate results Decreased use of reagents Less use of man power

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

29. http ://www.immunohistochemistry.us /

30. THE END ANY QUESTIONS