Specimens for IHC Flashcards
Specimen is prepared as cryostat section and must be fixed
Frozen Section
Frozen Section is carried out
Intra-operatively
Frozen Section is used for
RAPID DIAGNOSIS
Purpose of Fixation in IHC
- Preserve position of the antigens being detected
- Prevent destruction of labile antigenic sites
Microtomes used for Cutting specimen in IHC
- Freezing
- Cryostat
Fixative agent used for IHC Frozen Section specimen
- Methanol
- Acetone
2 Types of Processed Specimens
- Formalin-fixed
- Paraffin embedded
Process of retrieving antigens and transferring it into the glass slide
Antigen Retrieval
Most commonly used fixative in IHC
Formaldehyde
Method only done for formalin-fixed and paraffin embedded specimen
Pre-treatment blocking
2 Types of Pre-treatment blocking
- Peroxidase Blocking
- Protein Blocking
Blocks endogenous peroxidase
Peroxidase block
Substances that is being used for Peroxidase blocking
3% Hydrogen Peroxide
Type of pretreatment blocking that Blocks non-specific site and reduces background
Protein blocking/ Protein block
Protein block uses what substance
10% normal serum
Preferred pre-treatment blocking
Protein block
2 General Methods for Antigen Retrieval
- Heat Induced Epitope Retrieval
- Protease Induced Epitope Retrieval
Enzymes being used in PIER
- Trypsin
- Protease
Methods under HIER
- Microwave
- Pressure cooker
- Water bath, autoclave, and steamer heating
- Decloaker heating
- microwave and enzyme
- Digestion
Less time consuming HIER method
Pressure cooker
A HIER method that runs for 20 mins
Microwave
Purpose in the use of CONTROLS in IHC
- Test for specificity of antibodies involved
- Prevent misinterpretation of false (-) and false (+)
Control: Tissue with antigen
Positive control
Control: omitting primary antibody from staining or replacing primary antibody with IgG that is directed against an unrelated antigen
Negative Control
Also known as Built In control
Internal Tissue Control
Built In control contains
Target antigen in tissue elements and adjacent normal tissue elements
