Immunohistochemical Flashcards
Does fixation impact the ability to perform immunohistochemical staining?
Yes, many tissue antigens do not survive traditional fixation procedures and frozen sections need to be used, e.g. some lymphoid cell surface antigens.
Prolonged fixation time in 10% NBF reduces immunoreactivity in many antigens.
What does the term “masking” mean in immunohistochemical?
It refers to the blocking of antigen/epitope sites or “masking” by fixation in formaldehyde from the cross linkages that are formed.
Therefore, if formalin fixed, a pretreatment of the tissue is required to expose the antigen/epitope sites.
What are the methods of epitope retrieval that are used in immunohistochemical?
2 and #3 are kind of similar
- HIER - Heat induced Epitope Retrieval - use high heat combined with a liquid (e.g. buffer) to undo cross linkages with a pressure cooker, microwave, etc.
- EIER - Enzyme induced Epitope Retrieval - cleave proteins or break bonds at certain amino acids and unmasks the cross-link of certain antigens.
- PIER - Proteolytic Induced Epitope Retrieval
- Chemical unmasking - can use acids, bases, or other reagents.
What problems can occur with pre-treatments to unmask eptiopes?
Pre-treatment can expose potential sources of background that may require blocking to avoid / reduce false-positives or non-specific staining.
What are some general types of blockers in immunohistochem?
- Protein blockers
- Enzyme blockers
- Biotin blockers
- Fc blockers
Selection and application depends on the source of non-specific staining to be eliminated.
What do protein blockers do?
Protein blockers block non-specific charges on the tissues (+/-) that can attract the (+/-) charges on the antibody [not related to the specific Ab-Ag fit].
Con: Binding of weak or low affinity antibodies may be adversely affected.
What is the purpose of enzyme blockers?
Endogenous enzymes (enzymes originating w/in the cell) may react with the chromogen and substrate from the detection kits. Hence, they can stain even though the Ab does not.
What is an example of an endogenous enzyme that needs to be blocked when the detection kit uses horseradish peroxidase as the enzyme conjugate? What is it blocked with?
Endogenous peroxidase (found in blood cells, hemoglobin), tissue macrophages, liver, kidney.
Blocked with:
- 1-3% hydrogen peroxide,
- sodium azide or
- sodium azide in 3% hydrogen peroxide
- 3% hydrogen peroxide in methanol.
What can the endogenous enzyme alkaline phosphatase be blocked with and when needed? Where is it found?
Blocked with:
1. Levamisole (except GI tract)
2. Bouin’s (GI tract)
Needed when detection kit uses alkaline phosphatase as the enzyme conjugate.
Alkaline Phosphatase - mostly unique to kidney’s, placenta, prostate, and GI tract.
When does a biotin blocker need to be used?
Endogenous biotin found in liver, kidney, spleen, pancreas, and central nervous system. Also other locations but not as pronounced.
Biotin blocker is needed with kits employ avidin or strept-avidin as part of the conjugate. Problem is exacerbated by HIER.
Why are Fc blockers needed (Fc is blocking Abs)?
Some normal tissues and some disease states increase the production of antibodies. They diffuse out from source of production and bathe the surrounding tissues. Called diffuse, interstitial Ig. Blockers are used to stop these reacting with primary and secondary antibodies used in the kit though their Fc portions
What is ‘cross-reactivity’ in immunohistochem?
Cross reactivity can occur when the antigenic sites or the protein in question is shared by another protein.
How can ‘cross-reactivity’ in immunohistochem be blocked?
Not straight forward. Various techniques include:
- Use affinity purified antibodies
- Block with normal serum of same species as the primary antibody.
- Dilute the antibody further
- Use diluents with higher salt content or adjust the pH
Compare the difference between monoclonal Abs to polyclonal in their specificity and sensitivity? (generally)
What are characteristics of a “good” primary antibody?
