IHC Flashcards
What is the purpose of IHC methods in pathology?
Determine origin of tumors and determine between benign and malignancies.
What are considered normal results when using IHC methods?
- Tissue/cells appear normal and have high level of maturity
- Cells are appropriate to tissue type
- No specific tumour antigens present
What are advantages to monoclonal antibodies?
- No batch to batch variability
- Purer than polyclonal
- high affinity and specificity
How is IHC used in histology?
- Antibodies bind wherever antigens are present
- excess can be washed away
- Antibodies can have fluorescent labels on them for visual demonstration under the microscope
What are two epitope retrieval methods for IHC?
HEIR & EIER
What is the best preparation method for lymphocytic surface markers when doing IHC methods?
Cryostat
What are the advantages of epitope enhancement?
- Dilute antibodies further
- Expose epitope sites
- uniform staining
- intense reactions with decreased incubation
What are the two most common solutions used for HEIR method?
- Sodium citrate
- EDTA
What is the purpose of increasing the pH for HEIR method?
- enhances nuclear and cell surface antigens
What are the disadvantages of HEIR?
- Tissue damage when heating strong alkaline solutions
- Tissue detachment
- Holes in tissue section
What are the methods of heating HEIR slides?
- Microwave
- Pressure cooker
- Vegetable steamer
- Circulating Water bath
What is used in the EIER method to expose antigenic sites in tissue sections?
Proteolytic enzymes
What are the disadvantages of using EIER?
- Non specific staining if used improperly
- Can weaken specific staining
- Fragmentation or loss of tissue sections
- Over digestion - distorted tissue morphology
What is the purpose of combining HEIR and EIER?
- Minimize the extend of enzymatic pretreatment
When combining HEIR and EIER, what order are they done?
- HEIR
- EIER
What is a fluorochrome?
- Dye that absorbs light and emits it’s own light at longer wave lengths
What are the advantages of using fluorochromes?
- Sensitive
- Specific
- Simple
What is enzyme IHC?
Enzyme demonstrates antibody location in the presence of substrate and chromogens.
What are 4 enzymes used as markers?
- Alkaline phosphatase
- B-galactosidase
- Glucose oxidase
- Horseradish peroxidase
In tissue containing melanin what colour of chromogen should be used when doing enzyme IHC?
RED
What counter stain can be used for enzyme IHC methods? Why?
Mayer hematoxylin - bc it doesn’t contain alcohol
What would happen is Harris hematoxylin was used to counterstain a section undergoing enzyme IHC staining?
False negative results because ACE and AP are dissolved in alcoholic hematoxylins
What is the principle of direct immunofluorescent techniques?
Labeled antibody of known specificity is used to identify antigen’s in patient tissue, which are subsequently reacted with a chromogen.
What are advantages to direct IHC methods?
- One antibody
- Short and quick
What are the disadvantages of direct IHC methods?
- Little signal amplification
- Lacks sensitivity
- May not demonstrate low levels of antigens in tumours.
What is the principle of indirect immunofluorescent techniques?
- Unlabeled primary antibody reacts to tissue antigens
- Labeled secondary antibody reacts to the primary antibody.
Compared to the direct method, what are advantages of the indirect method of IHC?
- More sensitive
- Secondary antibody can usually bind with multiple different antibodies.
What is the purpose of using a third antibody in an indirect IHC method?
- Further amplify the signal
What is the light microscope method of IHC?
3 step method:
- Primary antibody
- Secondary antibody
- soluble enzyme-anti-enzyme complex
What is the source of the soluble enzyme in the light microscope method of IHC?
From the same source as the secondary antibody.
What is the PAP complex?
3 molecules of peroxidase and two antibodies.
What is the APAAP complex?
2 molecules of alkaline phosphatase and one antibody.
What are the two avidin- biotin methods?
- ABC (avidin-biotin complex method)
- LAB (Labeled avidin-biotin)
What is the principle of avidin?
Avidin irreversibly binds to biotin.
What are the steps in the avidin-biotin techniques?
- Primary antibody
- Biotinylated secondary antibody
- (ABC) preformed avidin-biotin enzyme complex
- (LAB) enzyme labeled avidin
What is the purpose of pretreating sections with peroxide.
Remove endogenous peroxidase enzyme activity, decreases non specific staining
What is a polymer enzyme?
secondary antibody fused with HRP or AP
What is the purpose of PAP?
Localize tissue antigens
What fixatives are best for Basic PAP?
NBF with epitope retrieval
What are positive reactions for basic PAP?
Brick red
What can you use to coverslip IHC slide with synthetic resin?
Crystal mount, unless using DAB instead AEC
What are DAB and AEC?
Chromogens
What are two methods of storing control slides?
- Store at -20C
- Dip pre-cut slides in paraffin, melt in over before use
What are advantages of using pre-diluted antibodies?
- Ready to use
- Optimal dilution by the vendor
What are disadvantages of using pre-diluted antibodies?
shelf life is shorter
What are advantages of using concentrated antibodies?
Have longer shelf life and can be snap frozen with liquid nitrogen.
What are disadvantages of using concentrated antibodies?
Require dilution to optimal concentration prior to use
What are three examples of appropriate containers for storing antibodies?
- Borosilicate
- Propylene
- Polycarbonate
What can be added to antibodies to prevent loss/ adsorption of proteins to the storage container?
Albumin
What are the two blocking reactions of immunoperoxidase method?
- peroxide in absolute methanol = block endogenous peroxidase activity.
- human serum - block non specific staining
