This is a presentation I prepared to demonstrate my mastery of the basics of Immunohistochemistry during my first two months of employment as a Biologist at the Cell Marque Corporation. Please note, there are a few slides that appear to be dysfunctional and overlapping; this is due to the fact that these particular slides included complex animations that I designed to illustrate various scientific concepts related to the practice of Immunohistochemistry. If you wish to view this presentation in its entirety (animations included), feel free to contact me via LinkedIn and I will gladly provide you with a fully-functional version.

1. Basics of
Immunohistochemistry
Jake Lyons

2. What We Will DiscussWhat We Will Discuss

3. Immuno histo chemistryImmuno histo chemistry
A Synergy of 3 Scientific Disciplines

4. “IHC is a biochemical assay technique that takes advantage
of chemical reactions to locate and visualize the interaction
between antibodies and their target antigen on sample
tissue”
What is Immunohistochemistry?What is Immunohistochemistry?

5. What We Will DiscussWhat We Will Discuss

6. IHC SignificanceIHC Significance
 While there are countless medical detection systems (x-ray, CT-
scan, MRI etc.); There are few identification tools available.
 With new carcinogens being discovered and an ever increasing
population; the demand for cancer diagnostics is at an all-time high.
 Even with the emergence of genetic testing; in many cases, IHC
remains the most cost-effective diagnostic tool from which
accurate and often life-saving diagnosis and/or prognosis can be
derived.

7. 2 Primary Applications2 Primary Applications
Diagnosis:
 Identification of the
cause of an illness or set
of symptoms
{Infectious Agent?}
Prognosis:
 Predicting the likely outcome of
an illness

8. Where is IHC Performed?Where is IHC Performed?
Clinical Labs
- Provide info on the health of the patient;
diagnosis/prognosis (Hospitals)
Reference Labs
- Large scale testing facility;
have extensive array of diagnostic
tools; receive toughest cases
Research labs
- Associated with
Universities; testing is
more experimental;
generate publications

9. What We Will DiscussWhat We Will Discuss

10. Immunology (immuno-)Immunology (immuno-)
Definition:
The branch of medicine
and biology concerned
with immunity.

11. The Immune SystemThe Immune System

12. AntibodiesAntibodies
Definition:
Large, Y-shaped proteins used by the
immune system to identify and neutralize
foreign objects (antigens).

13. Antibody Structure (IGG)Antibody Structure (IGG)

14. ““Y” Use Antibodies?Y” Use Antibodies?
IHC takes advantage of the following antibody attributes:
 Ability to locate and tag target proteins with incredible specificity
 Constant region that can be located by secondary antibodies
 Large molecules that can be outfitted with detection systems

15. AntigensAntigens
Definition:
- A toxin or other foreign substance that elicits an immune
response in the body (bacteria, viruses, tumors)

16. EpitopesEpitopes
The outer surfaces of antigens are covered by unique 3-
dimensional structures known as epitopes

17. Antigen RecognitionAntigen Recognition
Antibody variable region is highly attracted to specific epitope(s)

18. Immuno histo chemistryImmuno histo chemistry
A Synergy of 3 Scientific Disciplines

19. 2. Histology (histo-)2. Histology (histo-)
Definition:
The study of the microscopic structure of organic
tissues. {Can you guess the tissue type depicted below?}

20. From Biopsy to SlideFrom Biopsy to Slide
 Biopsy
 Fixation (10% NBF)
 Dehydration & Clearing
 Embedding
 Microtomy (3-4 microns)
 Apply tissue to (+) charged slide
 Baking (2-4 hours/overnight for fatty tissues)

21. Immuno histo chemistryImmuno histo chemistry
A Synergy of 3 Scientific Disciplines

22. 3. Chemistry3. Chemistry
Definition:
The science of the composition,
structure, properties and
reactions of matter
Examples of IHC Chemical Reactions:
 Binding of antibody to antigen
 Enzyme and chromogen interaction

23. Immuno histo chemistryImmuno histo chemistry
A Synergy of 3 Scientific Disciplines

24. What We Will DiscussWhat We Will Discuss

25. IHC in Practice: An OverviewIHC in Practice: An Overview

26. Quick Review of Histology…Quick Review of Histology…
 Biopsy
 Fixation (10% NBF)
 Dehydration & Clearing
 Embedding
 Microtomy (3-4 microns)
 Apply tissue to (+) charged slide
 Baking (2-4 hours/overnight for fatty tissues)

27. IHC in Practice: An OverviewIHC in Practice: An Overview

28. PretreatmentPretreatment
Objective: Return tissue to native state
3 Steps
Deparaffinization:
Removal of wax
Rehydration:
Using graded alcohols (decreasing concentration)
Epitope Retrieval:
Removal of formalin cross-links

29. PretreatmentPretreatment
Antigen SurfaceAntigen Surface
Epitopes

30. Summary of PretreatmentSummary of Pretreatment

31. IHC in Practice: An OverviewIHC in Practice: An Overview

32. Application of Primary AntibodyApplication of Primary Antibody
Antigen SurfaceAntigen Surface
Epitopes
Primary Antibody

33. IHC in Practice: An OverviewIHC in Practice: An Overview

34. Detection SystemsDetection Systems

35. LSABLSAB
Antigen SurfaceAntigen Surface
Epitopes
Primary Antibody
Secondary Antibody Biotinylated link
Streptavidin
Streptavidin
Enzymes
Chromogen
Visible Precipitate
Visible Precipitate

36. 1-Step Polymer1-Step Polymer
Antigen SurfaceAntigen Surface
Epitopes
Primary Antibody
Dextrin
Conjugated enzymes
Secondary Antibody
Chromogen
Visible Precipitate

37. 2-Step Polymer2-Step Polymer
Antigen SurfaceAntigen Surface
Epitopes
Primary Antibody
Secondary Antibody
(Amplifier)
Tertiary Antibody
(Detector)
Dextrin
Conjugated enzyme
Chromogen
Visible Precipitate

38. IHC in Practice: An OverviewIHC in Practice: An Overview

39. CounterstainCounterstain

40. IHC in Practice: An OverviewIHC in Practice: An Overview

41. CoverslippingCoverslipping
 After being counterstained, slides are now ready to be
coverslipped to preserve the stain and allow for viewing under
the microscope

42. What We Will DiscussWhat We Will Discuss

43. What We Will DiscussWhat We Will Discuss

44. Questions?Questions?