7. Immunohistochemistry

Question 1

What is immunohistochemistry?

Answer 1

Method using labelled antigens to identify and localise specific proteins

Consists of specific interaction b/w antigen and it’s antibody to identify specific proteins

-Proteins must be purified using biochemical /molecular methods  to produce antibody against it

Question 2

What is immunohistochemistry used for?

Answer 2

Diagnostic and Research proposes

To detect specific proteins / other molecules in cells/tissues

Question 3

Types of antibodies?

Answer 3

POLYCLONAL ANTIBODIES:
•Collection of immunoglobulin molecules that reacts against specific antigen – identifying a different epitope

MONOCLONAL ANTIBODIES:
•Antibodies made by identical immune cells

Question 4

What does the body do with antigens?

Answer 4

The body’s immune cells prod antibodies against antigens (macromolecules that are not a normal part of the organism - ‘foreign’) - can be dangerous

Question 4

How do you produce antibodies against a certain protein?

Answer 4

Isolated protein of one species is injected into animal of another species
If protein’s AA is suff different to be recognised = animal think it is antigen and prod antibodies

Question 5

What are antibodies normally tagged with?

Answer 5

• Fluorescent compounds
• Peroxidase
• Alkaline phosphate (histochemical detection)
• Electron dense gold particles (TEM)

Question 6

What happens in immunohistochemistry for tissue sections that has a protein of interest?

Answer 6

• incubated in a solution w/ labelled antibody to the protein
• the AB binds to protein (specifically)
• location can be seen either LM/EM depending on how antibody is labelled

Question 8

What family do antibodies belong to?

Answer 8

Belong to the immunoglobulin family of glyproteins - produced by lymphocytes

Question 9

What is the indirect method of immunohistochemistry?

Answer 9

Indirect Method:

• More sensitive method – requires 2 antibodies
• Instead of labelling primary antibody spec to protein ‘x’ – the tag is conjugated to secondary antibody
• The secondary antibody is made in a different foreign species against the immunoglobulin class (where primary antibody belongs to)

Question 10

What is the direct method of immunohistochemistry?

Answer 10

Direct Method:

• The antibody (monoclonal/polyclonal) is tagged itself w/ label
• Tissue section is incubated w/ antibody for some time = so antibody interacts/binds to protein ‘x’
• The section is washed to remove unbound antibody
• Processed by appropriate method and examine microscopically to study location/other things about protein ‘x’

Question 11

What is an epitope?

Answer 11

The part of an antigen to which antibody attaches itself