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

15. EpitopesEpitopes

16. Antigen Epitope InteractionAntigen Epitope Interaction
Antibody variable region is highly attracted to specific epitope(s)

17. ANTIBODY PRODUCTIONANTIBODY PRODUCTION
Mature B-
cell
Antigen
Helper T-
cell
Memory
cell
Plasma cell

18. Antibody Production Using a HostAntibody Production Using a Host

19. Monoclonal AntibodiesMonoclonal Antibodies
Specific to only one epitope on a particular antigen
Produced by B cells that are clones of one another
(descended from a single parent cell)
Advantages:
 Offer SPECIFICITY resulting in precise staining
signal
 Can be combined with other monoclonals to create
cocktails

20. Mouse Monoclonal Antibody GenerationMouse Monoclonal Antibody Generation
Antigen
injection
Suspension
of B
lymphocyte
s isolated
from spleen
Cultured
myeloma
cells
Suspension
of myeloma
cells
Cells fused to create
hybridomas

21. Mouse Monoclonal Antibody ProductionMouse Monoclonal Antibody Production
Individual fused cells are separated for
culturing
Successfully transformed hybridoma
cell lines proliferate
Each hybridoma culture is monoclonal: will
generate a specific clone

22. Polyclonal AntibodiesPolyclonal Antibodies
Specific to multiple epitopes on a single antigen
Produced by multiple B cell clones (multiple parent
cells that each give rise to their own antibody clone)
Advantages:
Offer SENSITIVITY resulting in stronger staining signal

23. Rabbit Polyclonal Antibody ProductionRabbit Polyclonal Antibody Production
1. Immunization
with antigen
 Antigen
injected into
bloodstream
to elicit
immune
response
 Subsequent
monthly
injections
2. Antibody
production
B cells secrete
multiple
antibody clones
for various
epitopes on the
injected antigen
3. Serum
retrieval
 Serum
harvested
from
rabbit’s ear
4. Serum
purification
 Serum
filtered to
separate
antibodies
from
remaining
fluid

24. Rabbit Monoclonal Antibody ProductionRabbit Monoclonal Antibody Production
Newer technology
that combines the
specificity and
consistency of
mouse
monoclonals with
the diverse epitope
recognition of rabbit
polyclonals

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

26. What We Will DiscussWhat We Will Discuss

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

28. 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)

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

30. What We Will DiscussWhat We Will Discuss

31. ChemistryChemistry
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

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

33. What We Will DiscussWhat We Will Discuss

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

35. 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)

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

37. 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

38. PretreatmentPretreatment
Antigen SurfaceAntigen Surface
Epitopes

39. Summary of PretreatmentSummary of Pretreatment

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

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

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

43. Detection SystemsDetection Systems

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

45. 1-Step Polymer1-Step Polymer
Antigen SurfaceAntigen Surface
Epitopes
Primary Antibody
Dextrin
Conjugated enzymes
Secondary Antibody
Chromogen
Visible Precipitate

46. 2-Step Polymer2-Step Polymer
Antigen SurfaceAntigen Surface
Epitopes
Primary Antibody
Secondary Antibody
(Amplifier)
Tertiary Antibody
(Detector)
Dextran
Conjugated enzyme
Chromogen
Visible Precipitate

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

48. CounterstainCounterstain

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

50. CoverslippingCoverslipping
 After being counterstained, slides undergo dehydration and
clearing before being coverslipped to preserve the stain and allow
for viewing under the microscope

52. Questions?Questions?