Immunohistochemistry principle Flashcards
What does ABC stand for in the context of antibody detection methods?
Avidin-biotin complex
ABC method is a 3-step antibody detection method that employs an indirect technique.
What is the role of cytokeratin in immunohistochemistry?
Antigen on epithelial cells
What does CD45 represent in immunology?
Antigen on leucocytes
What is the first step in the ABC method?
Pretreated with enzyme or heat to enhance antigenicity masked by chemical fixatives
What is the purpose of treating with hydrogen peroxide in the ABC method?
Blocks activity of endogenous peroxidase present in blood cells
What does normal serum do in the ABC method?
Binds to non-specific epitopes to prevent non-specific binding
What binds specifically to the target antigen in the ABC method?
Primary antibody
What type of secondary antibody is used in the ABC method?
Biotinylated secondary antibody
It can be either rabbit anti-mouse or swine anti-rabbit Ig.
What is the specificity of avidin in the ABC method?
Highly specific for biotin
What is avidin conjugated to in the ABC method?
Horseradish peroxidase (HRP) enzyme
What does the DAB reaction produce in the ABC method?
Brown polymer
The peroxidase on ABC acts on substrate hydrogen peroxide, releasing oxygen and water, which reacts with DAB.
What is the visual outcome of the antigen-antibody reaction in the ABC method?
Brown deposit
What is the property of Dakopen in immunohistochemistry?
Hydrophobic, confines liquid inside
What are chromogens used for in immunohistochemistry?
Chemical compounds that produce a coloured product visible by light microscopy
What is the purpose of PBS in immunohistochemistry?
Used as a washing buffer to remove excess antibodies and reagents while stabilizing antigen-antibody binding
