Following incubation with the primary antibody, antibody binding is visualized using an appropriate detection system. The method of detection can be direct or indirect, and may generate a fluorescent or chromogenic signal. Direct detection involves the use of primary antibodies that are directly conjugated to a label. Indirect detection methods utilize a labeled secondary antibody raised against the primary antibody host species. Indirect methods can also include amplification steps to increase signal intensity. Commonly used labels for the visualization of epitope-antibody interactions include fluorophores and enzymes that convert soluble substrates into insoluble, chromogenic end products. The choice of label is affected by the detection method employed, personal preference, and the type microscope equipment that is available
Direct or Indirect Detection & Amplification
The choice of whether to use direct or indirect detection is often dictated by the level of antigen expression. For example, detection of a highly expressed epitope might be possible using a primary antibody directly conjugated to a label. Advantages of direct detection include the ease of use for multicolor staining and the elimination of concerns regarding non-specific binding of the secondary antibody. The major disadvantage is that direct detection may lack the sensitivity required to visualize lower expression levels. In rare cases the conjugation process may also have a negative impact on the affinity of the primary antibody.
In contrast, indirect detection methods generally have a higher level of sensitivity and generate a more intense signal. The signal is amplified using the indirect method because of the potential for at least two labeled secondary antibodies to bind to each primary antibody. However, the use of a secondary antibody requires extra blocking steps and additional controls.
Further amplification of the signal can be achieved by taking advantage of the strong affinity of avidin and streptavidin to bind biotin. Avidin is a glycoprotein in egg white that combines stoichiometrically with biotin. Streptavidin is purified from the bacterium Streptomyces avidinii, is not glycosylated, and exhibits lower non-specific binding than avidin. Both proteins bind four biotins per molecule. If a biotinylated secondary antibody is employed, the signal can be significantly amplified by subsequent incubation with an avidin-biotin complex (ABC Method), or labeled streptavidin-biotin (LSAB Method). Streptavidin may be conjugated to a detection enzyme (i.e. horseradish peroxidase or alkaline phosphatase) or fluorochrome. Using these amplification methods requires additional steps to prevent non-specific binding (see Preventing Non-specific Staining).
Detection Methods & Signal Intensity
Fluorescence detection is based on the use of fluorochromes that emit light when excited by light of a shorter wavelength. The fluorochrome can be conjugated directly to the primary or secondary antibody, or to streptavidin. Immunofluorescence is commonly used for the simultaneous visualization of multiple cellular targets. For example, tissues can be incubated with a mixture of different primary antibodies followed by incubation with secondary reagents conjugated to fluorochromes that emit light at different wavelengths.
|Multicolor Fluorescence Microscopy. A typical immunofluorescence setup that depicts the light source, a suitable filter set for the fluorochrome of interest, and a detection mechanism.
Multicolor experiments must be designed to limit cross-reactivity between the detection reagents and crossover between the spectral properties of the fluorochromes being used. To limit cross-reactivity between secondary reagents, the primary antibodies should be derived from different species. This allows for the use of species-specific secondary antibodies that recognize only one primary antibody. However, there may be some instances when it is necessary to simultaneously use antibodies from the same species. This can be achieved using a biotinylated form of one of the primary antibodies. When using this technique, tissues must be incubated with the non-biotinylated antibody first, followed by incubation with the fluorophore-conjugated secondary antibody. The tissues are then incubated with the biotinylated primary antibody followed by incubation with a streptavidin-conjugated fluorochrome. This approach will ensure that the streptavidin conjugate will only bind the biotinylated antibody, limiting cross-reactivity.
|R&D Systems NorthernLights Fluorochromes have Non-overlapping Spectral Properties that make them Ideal for Multicolor IHC and ICC. A. Emission curves for NorthernLights-493 anti-goat IgG (Catalog # NL003; green) and NorthernLights-557 anti-mouse IgG (Catalog # NL007; red). B. Multicolor detection of Nestin and beta-III Tubulin in rat cortical stem cells (Catalog # NSC001) using anti-rat Nestin affinity-purified polyclonal antibody (Catalog # AF2736; green) and anti-neuron specific beta-III Tubulin monoclonal (clone TuJ-1) antibody (Catalog # MAB1195; red). Cells were stained with NorthernLights-493 (Catalog # NL003; green) and NorthernLights-557 (Catalog # NL007; red) secondary antibodies and counterstained with DAPI (blue).
To facilitate chromogenic detection, the primary antibody, secondary antibody, or streptavidin is conjugated to an enzyme. When a soluble organic substrate is applied, the enzyme reacts with the substrate to generate an insoluble colored product that is localized to the sites of antigen expression. Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase, which convert 3,3' diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), into brown and red end products, respectively. DAB is more commonly used than AEC since the latter is soluble in alcohol and is more prone to fade when exposed to excessive light. Aqueous counterstain and mounting media must be used with AEC.
Chromogenic detection is considered to be more sensitive than that of immunofluorescence, but is less convenient because it includes more incubation and blocking steps. Like immunofluorescence, chromogenic detection allows for the visualization of multiple antigens, but only if the antigens are confined to different locations in the cell and tissue because overlapping colors may obscure results. An advantage of DAB chromogenic staining is that the colored precipitate formed during the reaction between HRP and DAB is not sensitive to light and the slides can be stored for many years. Unlike fluorescence microscopy, which requires a specialized light source and filter sets, chromogenic techniques require only a typical light microscope.