Immunohistochemistry new

1. Immunohistochemistry
Spring 2014
General Immunology
The immune system exerts its control
through humoral ( antibody) and
cellular components.

2. Antibody
• What is an antibody? An antibody is a hhoosstt pprrootteeiinn
( immunoglobulin) produced in response to the presence
of foreign molecules, organisms, or other agents in the
body.
• Antibodies commonly known as immunoglobulins are
proteins that are produced by B- lymphocytes in
response to antigenic stimulation.
• An immunoglobulin is aa YY-- sshhaappeedd pprrootteeiinn molecule that
is composed of both heavy and light chains.

3. Antibody classification
• Classes of antibody differ in structure and function with
each immunoglobulin antigenically distinct.
• An immunoglobulin is composed of 2 identical heavy
chains α, γ,ε,μ, or delta, which determine the
immunoglobulin subclass ( Ig A, IgG, Ig E, Ig M or Ig D)
• The light chain is either κ or λ. These proteins are
expressed on the cell surface or membrane, and
secreted into the blood and other fluids by plasma cells.
• The upper arm of the Y are the regions of the antibody
that bind to the short arms of their specific antigen.

4. Antibody classification Contd.
• Epitope- the site at which the antibody
attaches to the tissue.
• An antibody may target more than 1
antigen but is specific for for only 1
epitope.
• Proteins are very good antigens because
of their large size. The fact that anibodies
( immunoglobulins ) are proteins makes
them very potent antigens.

5. Antigen
• What is an antigen? An antigen is a molecule made up of proteins,
carbohydrate or other polymers, and is capable of producing an
immune response in animals or cell cultures for the production of
antibodies.
• Antigens in tissues bind antibodies at different cell sites,: the
membrane, cytoplasm, nucleus, organelles or a combination of
more than 1.
• Many compounds are antigenic.
• Because of their large size proteins are ideal for immunization and
for binding but polysaccharides, nucleic acids, and other polymers
can also be antigenic.
• The most common antigen that induce antibody production are
bacteria and viruses.

6. Polyclonal antisera
• Pooling of antibodies to make polyclonal antisera
• Blood drawn from human arm, isolate IIgg MM aannttiibbooddyy
from it which is injected into a rabbit. The human Ig M
antibody would act as an antigen and stimulate the rabbit
to make Anti-human Ig M antibody. Serum from the
immunized rabbit could be used as a polyclonal anti-human
Ig M antibody reagent.
• Polyclonal antiserum is highly sensitive because it binds
to more than 1 epitope, but is not as selective and gives
non specific staining background.
• Examples of hosts that are used to produce polyclonal
antisera include rabbit, goat, horse, sheep and
humans.

7. Monoclonal antibodies
• Monoclonal antibodies (MABs) How are they prepared?
• MABs- prepared by injecting mice with an antigen.
• B-lymphocytes are fused with nonsecreting myeloma
cells ( non secreting plasma cell tumor) MABs can be
characterized, standardized, and produced in unlimited
quantities. Mouse MABs are purer than polyclonal
antibodies. MABs have high affinity and selectivity.

8. Immunofluorescence
• What is immunofluorescence ?
• Imf- the oldest pathology immunohistochemical technique.
• Fluorochrome- dye that absorbs light and then emits iittss oowwnn lliigghhtt
at a longer wavelength. This phenomenon of absorption and
emission of light is called fluorescence.
• Fluorochrome attached or conjugated to antibody, the sites of
reaction between antigen and labeled antibody can be visualized
easily.
• Commonly used fluorochrome in immunofluofluorescence technique
are fluoescein isothiocyanate ( FITC) and rhodamine.
• Both of these dyes absorb light that is not visible to the human eye.
(UV) and emit light that is visible.

9. Invasive Carcinoma of the
Breast
Chromogen used akloline phosphatase

10. Breast Carcinoma
Her 2 Dab Chromogen

11. Immunohistochemistry Questions
Define the following
1. Antigen
2. Antibody
3. Chromogen
4. Epitope
5. For what purpose is immunoistochemistry used?
6. What type of compound is antigenic?
7. What common antigens induce antibody production?
8. Define immunoglobin
9. Define Polyclonal Antisera
10.Define Monoclonal
11.Which antibodies are purer?
12.How long should breast tissue be fixed for IHC?
13.Define Fluorochrom
14.What 2 frozen section tissue are routinely examined with
immunoflourescence?
15.Define Direct Method
16.Define Indirect Method

12. Enzyme Immunohistochemistry
Questions
15. What enzmes are used in enzyme immmunohistochemistry?
16. What are the most common enzymes chosen for antibody
visualization?
17. List property of AEC
18. List property of DAB