Practical 7: Immunohistochemistry Staining Flashcards
What method of immunohistochemistry staining was used?
Avidin-Biotin Complex (ABC)
Explain what the Avidin-Biotin Complex is
A three step antibody detection method
Whereby an antibody is labelled with peroxidase enzyme is applied to visualise antigenic sites
What are the three main uses of immunohistochemistry?
ID of immune cells
ID of tumour cells
ID of micro-organisms
What must be done to slides prior to beginning immohistochemistry?
Antigen retrieval must be carried out
What two antigens were examined with immunohistochemistry?
Cytokeratin 19 -> CK19
CD45 (Common leucocyte antigen)
Where is cytokeratin found?
Epithelial tissue
Where is CD45 found
In all leucocytes
What are two ways by which antigen retrieval can be done?
Enzyme treatment
Heat treatment
Why does antigen retrieval need to be done?
To enhance antigenicity masked by chemical fixatives
Explain the principle behind the avidin biotin method of immunohistochemistry.
(7)
Primary antibody is an anti-human antibody (e.g. a mouse or rabbit anti-human antibody) which is specific for the antigen
Secondary antibody is an antibody against the primary antibody e.g. mouse anti-rabbit antibody
Avidin and biotin have a high affinity for each other
Avidin biotin complex consists of an avidin molecule with 3/4 binding sites bound to peroxidase-labelled biotin
The 4th binding site of avidin binds to the biotinylated secondary antibody
Oxygen and water are released -> the oxygen then acts on DAB and oxidises it to form a brown polymer
Why do you ring your slides with Dakopen?
This creates a hydrophobic ring which stops expensive stains and antibody reagents spilling off tissue
Minimises amount of stain used
Why must we use hydrogen peroxide
Stops peroxidase enzymes from working
This prevents background staining
This quenches endogenous peroxidase enzyme present in blood cells
