case 4 - IHC Flashcards
1
Q
IHC
A
Immunohistochemistry (IHC) is a laboratory technique used to visualize the presence and distribution of specific proteins or antigens within tissue samples. It combines principles of both immunology and histology to enable researchers and pathologists to study the location and abundance of proteins in cells and tissues
2
Q
procedure
A
- euthanasia
- tissue retrieval
- embedding
- sectioning
- slide prep
- antigen retrieval
- blocking
- primary Ab
- washing
- secondary Ab.
- washing
- detection (if needed)
- mounting
- microscopy
3
Q
primary Ab
A
- Recognition of Target Antigen: Primary antibodies are designed to specifically recognize and bind to the target protein or antigen of interest. These antibodies are generated through immunization of animals (e.g., rabbits, mice) with the target antigen or through recombinant DNA technology.
- First Binding Event: In IHC or IF, primary antibodies are applied to the sample first. They attach to the antigen within the tissue or cell section.
- Direct or Indirect Detection: Primary antibodies can be used for direct detection or in an indirect detection system. In direct detection, primary antibodies are directly conjugated to a detectable label, such as a fluorescent dye or an enzyme (e.g., horseradish peroxidase, HRP).
- Advantages: Primary antibodies are highly specific for the target antigen, making them essential for accurate detection. They are the critical reagents that determine what you are specifically looking for in your sample.
4
Q
secondary Ab
A
- Amplification of Signal: Secondary antibodies are not specific to the target antigen itself; instead, they are antibodies raised against the host species of the primary antibody. For example, if your primary antibody is from a rabbit, you would typically use an anti-rabbit secondary antibody. These secondary antibodies are conjugated to a detectable label.
- Second Binding Event: Secondary antibodies are applied after the primary antibody. They bind specifically to the Fc (constant) region of the primary antibody, creating a bridge between the primary antibody and the detectable label.
- Signal Amplification: Secondary antibodies serve to amplify the signal generated by the primary antibody. Since multiple secondary antibodies can bind to a single primary antibody, this amplification step enhances the detectability of the target antigen.
- Host Species Specific: Secondary antibodies are available for various host species, allowing flexibility when using primary antibodies raised in different animals.
5
Q
monoclonal Ab
A
- Origin: Monoclonal antibodies are derived from a single clone of B cells, which are identical in terms of their antigen specificity.
- Specificity: Monoclonal antibodies are highly specific. They recognize and bind to a single epitope (a specific part of an antigen). This means they target a single protein or antigen with precision.
- Production: Monoclonal antibodies are produced through hybridoma technology. A single B cell is fused with a myeloma cell (a cancer cell) to create a hybrid cell line that continually produces identical antibodies. This allows for large-scale production of a single type of antibody.
- Consistency: Monoclonal antibodies are consistent in their specificity and properties because they are derived from a single B cell clone. This consistency is advantageous in applications where precision is crucial.
- Applications: Monoclonal antibodies are commonly used in diagnostics, targeted therapies, and research. They are often used to treat specific diseases, such as some types of cancer or autoimmune disorders.
6
Q
polyclonal Ab
A
- Origin: Polyclonal antibodies are derived from multiple clones of B cells, each producing antibodies with varying specificities. They are generated by exposing an animal (e.g., rabbit, mouse) to an antigen, which triggers the production of antibodies from many different B cell clones.
- Specificity: Polyclonal antibodies are less specific compared to monoclonal antibodies. They can recognize multiple epitopes on an antigen, resulting in a mixture of antibodies with varying specificities.
- Variability: Polyclonal antibodies can vary from one batch to another and even from one animal to another because they are a mixture of antibodies from different B cell clones.
- Applications: Polyclonal antibodies are often used in research, particularly for their ability to detect a wide range of epitopes on an antigen. They are used in techniques like Western blotting and immunohistochemistry.
7
Q
detection methods
A
enzyme-based
fluorescent
chromogenic substrate-based
colloidal gold/silver amplification
tyramide signal amplification
8
Q
enzym-based detection
A
- Horseradish Peroxidase (HRP) and 3,3’-Diaminobenzidine (DAB): In this widely used method, a secondary antibody conjugated to HRP is applied to the tissue sections. When a substrate solution containing DAB is added, HRP catalyses the conversion of DAB to a brown precipitate at the site of antigen-antibody binding.
9
Q
fluorescent detection
A
- Direct Fluorescence: In this method, primary antibodies are directly conjugated to fluorophores (e.g., FITC, Texas Red, or Alexa Fluor dyes). The labelled primary antibodies are applied to the tissue, and fluorescence microscopy is used to visualize the signal.
- Indirect Fluorescence: Secondary antibodies that are conjugated to fluorophores are applied to amplify the signal. This technique is often used for multiple labelling experiments, as different secondary antibodies can be labelled with different fluorophores for simultaneous detection of multiple antigens.
10
Q
chromogenic substrate detection
A
- Avidin-Biotin Complex (ABC) Method: Biotinylated secondary antibody is used to bind to the primary antibody. The tissue is then treated with avidin-biotin-peroxidase complex, followed by addition of DAB substrate to produce a brown colour at the antigen-antibody binding sites.
11
Q
colloidal gold/silver ampl.
A
colloidal gold particles conjugated to secondary antibodies are used to bind to the primary antibody. After binding, silver enhancement reagents are applied, which deposit silver ions on the gold particles. The silver ions form metallic silver, making the target antigen visible under electron microscopy.
