IHC in Histopath, IMF in Practice

Question 1

What is immunohistochemistry used for?

Answer 1

• localising specific antigens in both tissues and cells based on antibody-antigen recognition.

Question 2

Immunohistochemistry methods

Answer 2

• One step direct
• Multiple step peroxidase - anti peroxidase (PAP)
• Avidin-biotin conjugate and Polymer based labelling systems were employed

Question 3

What were these methods used for?

Answer 3

• increase sensitivity and create clear visual results using light microscopy

Question 4

What are these methods performed on?

Answer 4

• originally performed on frozen sections it was swiftly applied to paraffin processed tissue

Question 5

Retrieval methods

Answer 5

• Enzyme digestion: “unmask” antigens that were altered by formalin fixation but was found not to improve IHC of majority of antigens.
• Optimal digestion times were also found to be hard to control.
• Antigen retrieval based on heating at high temperatures (Heat Induced Epitope Retrieval/HIER) was then introduced increasing the intensity of IHC dramatically

Question 6

Antibodies

Answer 6

• binds with a 2nd molecule specifically to an antigen
• production is induced specifically by the presence of the antigen.
• basic immune response.
• Ag is foreign to host cell, provoking immune response and Ab production

Question 7

Epitope

Answer 7

• cluster of Amino Acid residues on the antigen that will bind specifically to an antibody

Question 8

Monoclonal antibodies

Answer 8

• raised against specific antigens but don’t guarantee antigen specificity as other antigens may have similar epitopes
• However the “practical” specificity reflected by IHC is excellent.

Question 9

Polyclonal antibodies

Answer 9

• anti-serum containing several Ab’s having varying specificity against different antigens
• also include Ab’s to a range of bacteria/viruses that the animal may have encountered before it was used to source the Ab
• has the ability to cause non-specific background staining.
• polyclonal Ab’s are more sensitive but less specific than monoclonal Ab’s

Question 10

Background staining is caused by:

Answer 10

• Non-specific antibody/antigen binding
• Endogenous enzymes
• Peroxidase activity
• Endogenous biotin - present in RBC’s, WBC’s, eosinophils and hepatocytes. Peroxidase blocking is recommended in the form of H2O2 in Methanol
• Blocking of endogenous activity must be carried out before antibody application or the enzyme label would also be inactivated by blocking procedure