EPITOPE ENHAMCEMENT Flashcards
What is the most common fixation/processing protocol for IHC?
Formalin fixed, paraffin embedded (FFPE)
What is the major drawback of FFPE tissue?
Formalin induced genetic modification of the tissue can cause may result in the loss of antigen and antibody interaction
How does Heat-induced Epitope Retrieval (HIER) work?
Disrupting the formalin crosslinks by heating up to 100C or by a strong alkaline solution
What are the two common categories of epitope retrieval methods?
Heat (HIER)
Enzymatic (EIER)
When is epitope retrieval commonly used?
To break down hydrogen bonds that are formed during formalin fixation in tissue
What are the 7 advantages of epitope retrieval?
- Ability to further dilute antibody
- Exposure of epitope sites that were not previously detectable
- More intense reactions with decreased incubation times
- More uniform staining
- Decreased background staining
- Day-to-day staining consistency
- Improved standardization
What is the most important component of HIER?
Composition
pH
What are the major factors affecting epitope retrieval?
- pH
- Volume of the retrieval solution
- Heating time
- Temperature
When would a combination of EIER and HIER be used?
If the usage of an enzyme digestion alone reduces the quality of tissue due to a lengthy exposure time
How would a combination of EIER and HIER be used?
HIER is performed first, followed by rinsing and finally an enzyme digestion for a short amount of time (1-5 minutes)
How is imidazole used in the PAP technique?
Intensifies DAB reaction
What linking antibody would follow a monoclonal kappa primary antibody
Anti-mouse
Anti-rabbit
What do negative control slides omit?
Primary antibody
Uses nonimmune serum instead
What are the strong bonds called that form between proteins and calcium ions as a result of formalin fixation?
What type of epitope retieval is proteolytic-induced epitope retrieval (PIER)?
What two major factors require stringent control when performing proteolytic-induced epitope retrieval (PIER) antigen unmasking?
How does the epitope retrieval affect the choice of staining method and the staining result?
What is the mechanism of retrieval for EIER?