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
