Welcome

1. Welcome Introduction to IHC A basic overview of theory and techniques relating to immunohistochemistry • History • Theory • Techniques • Questions and Answers

2. IHC History • The principle of immunohistochemistry has existed since the 1930s • Started in 1941 with Coons identifying pneumococci using a direct fluorescent method. • Single antibody labeled with FITC molecule for visualization • Next came the indirect method, the addition of horseradish peroxidase, the peroxidase anti-peroxidase technique of 1979 and the use of the Avidin and Biotin complex in the early 1980s. • Methods utilizing enzyme conjugated antibodies were developed independently by Nakane and Pierce and Avrameas and Uriel

3. IHC Theory • Fixation • Cutting • Eptitope Retrieval • Primary Antibody • Secondary Labeling • Enzyme Reaction • Substrate Labeling

4. IHC Theory • Fixation • Stops the autolysis of proteins and maintains the properties of the tissue • Cross-Links Proteins with the cell • Essentially stops the meat from rotting • Should be done as soon as possible preferably in the Operating Room with an adequate amount of fixative

5. IHC Theory • Cutting • Performed on a microtome • Only well-fixed specimen • Charged slides recommended (VMSI instruments requires this for optimal results)

6. IHC Theory • Eptitope Retrieval • Heat Induced (HIER) • Uses physical heat to uncover cross-linked antigens • In theory, water will achieve this but there are better more specific buffers available • Protoelytic Induced (PIER) • Uses chemical enzyme to cut proteins • Pronase, Trypsin, Protinase K are all examples of enzymes that will achieve specific results • General protease works well most of the time

7. IHC Theory • Primary Antibody • 2 animals predominantly used in antibody production • Rabbit and Mouse until recently rabbit antibodies were polyclonal and mouse antibodies were monoclonal • Rabbit as polyclonal antibody production • Cells of interest injected into rabbit (e.g. skin for keratin antibody) • Immune response takes place • Rabbit blood serum is harvested and purified to obtain a “clean” antibody • Very sensitive antibody is created

8. IHC Theory • Primary Antibody • Mouse as monoclonal antibody production • A monoclonal antibody is a preparation of antibody that all have the same exact specificity, and in fact, is all the identical protein • one immunizes an animal, removes their spleen and purifies the B-cells that make antibodies. • These cells are then separated from one another, and each are fused to another cell that confers the ability to replicate and survive outside of the animal. (hybridoma) • The culture supernatants of these hybrid cells are tested for the antibody that you want. Once you find a hybrid cell making the antibody you want, you can grow as much of the now monoclonal antibody as you need. • This creates an antibody with great specificity

9. IHC Theory • Secondary Labeling • Typically an antibody derived from goat • Directed against the type of antibody used for your primary reaction i.e. anti-mouse goat or anti-rabbit goat • May be have many different labels • The labels include biotin, fluorescein, rhodamine, horseradish peroxidase and calf intestinal alkaline phosphatase.

10. IHC Theory • Enzyme Reaction – Avidin Binding Complex

11. IHC Theory • Enzyme Reaction – Labeled Avidin

12. IHC Theory • Substrate Labeling • Immunoenzymatic staining of tissues results from the reaction of a soluble substrate with an enzyme to produce an insoluble, colored product. • The intensity of the color produced when the substrate is added should correlate to the concentration of the primary antibody and the respective tissue antigen • The most common selections are horseradish peroxidase and calf intestinal alkaline phosphatase.

13. Questions?

14. IHC Techniques • Fixation • Cutting • Eptitope Retrieval • Primary Antibody • Secondary Labeling • Enzyme Reaction • Substrate Labeling

15. IHC Techniques • Fixation • As described before optimal fixation occurs if the specimen is placed into fixative immediately • Industry standard is 10% Neutral Buffered Formalin • Specimens should be fixed on processor a minimum of six hours to allow for adequate fixation • In order to maintain quality this practice should be as standardized as possible

16. IHC Techniques • Cutting • Well fixed specimens cut the best • For immunohistochemistry to work optimally cutting should be done between three and six microns • A sharp cutting blade helps eliminate knife chatter on the specimen. • Slides stain best when applied to charged slides • Slides have either a chemically or physically applied charge to the surface. This helps the slightly negatively charged specimen adhere to the glass.

17. IHC Techniques • Eptitope Retrieval - HIER • A method in which the cross linkage of proteins that occurs during fixation is undone • May be performed in a decloaker (pressure cooker), steamer, or in the microwave • Buffers of varying pH are available to optimize different antibodies • Cooking of the tissue varies from 10 to 60 minutes depending on the method used • The temperature achieved by these methods appears to be the critical variable. • Standardization at this step is the key to successful staining • Shi S-R, Cote C, Kalra KL, Taylor CR, Tandon AK (1992a) A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry. J Histochem Cytochem 40:787-792[Abstract]

18. IHC Techniques • Eptitope Retrieval – PIER • A method in which the cross linkage of proteins that occurs during fixation are cut • Protein specific proteases are available for this as well as general proteases • The difference between the two is the proteins they target • Excessive time in protease will start to break down proteins destroying the morphology of the cell

19. IHC Techniques • Primary Antibody • Applied directly to the specimen • Applied for 4 minutes to as long as overnight • Should be performed in a humidity chamber to avoid evaporation of antibody

20. IHC Techniques • Secondary Labeling • In actuality this is simply an antibody directed against a mouse or rabbit epitope • Applied directly to the specimen • Typical incubation is 10 to 30 minutes

21. IHC Techniques • Enzyme Reaction – ABC Method • Staining intensity is a function of the enzyme activity and improved sensitivity can be achieved by increasing the number of enzyme molecules bound to the tissue. The multiple binding sites between the tetravalent avidin and biotinylated antibodies (bound to the antigen) are ideal for achieving this amplification. The avidin-biotin complex (ABC) method was developed by Hsu and colleagues. A biotinylated enzyme (HRP or AP) is pre-incubated with avidin, forming large complexes to be incubated with the biotinylated antibody. Typically, the avidin and biotinylated enzyme are mixed together in a specified ratio for about 15 minutes at room temperature to form the complex. An aliquot of this solution is then added to the tissue, and any remaining biotin-binding sites on the avidin bind to the biotinylated antibody that is already bound to the tissue. The result is a greater concentration of enzyme (3 enzyme molecules to one avidin molecule) at the antigenic site and therefore an increase in signal intensity and sensitivity

22. IHC Techniques • Enzyme Reaction – LAB Method • If the avidin-biotin complex becomes too large to penetrate the tissue, a second method developed by the Guesdon and colleagues can be used. The labeled avidin-biotin (LAB) method employs an avidin-enzyme conjugate (or streptavidin-enzyme conjugate) to detect the bound biotinylated primary antibody on the tissue section. This smaller complex allow better tissue penetration. This is labeled with horseradish peroxidase or calf intestinal alkaline phosphatase.

23. IHC Techniques • Substrate Labeling • An insoluble colored end product that allows visualization of the chemical reaction that was performed on the tissue. • DAB (diaminobenzamidine) and a red or blue substrate for calf intestinal alkaline phosphatase • Other reagents may be added to enhance the color e.g. copper or nickel for DAB

24. Questions?

25. Current Trends in IHC • Routine surgicals • Imaging • New Molecular Tests

26. Current Trends in IHC • Routine surgicals • Routine surgical biopsies are being done more and more as an outpatient procedure removed from the hospital completely • These specimens are then transported to an off-site location for processing and interpretation • Typically done on prostate and skin samples • It appears that specialized testing will soon be the heart of the hospital laboratories and routines will be handled strictly on an outpatient basis

27. Current Trends in IHC • Imaging • Imaging of specimens is increasing at most large institutions • This allows for digital images of the specimen to be analyzed by an algorithm and assist the pathologist in interpretation • This does not take the place of the physician but gives them another tool to make a diagnosis • Also in the future may allow for collaboration around the world or around the corner

28. Current Trends in IHC • New Molecular Tests • With few exceptions most of the testing done in the IHC lab is based upon proteins and their expression • As drugs are developed that target specific genes rather than proteins new molecular test will need to be developed • The best example of this currently is FISH for Her2neu • This tests enables the pathologist to diagnosis an equivocal to either a positive or negative interpretation

29. Goodbye!