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Understand the difference between direct and indirect methods for immunofluorescence.

Immunofluorescence (IF) or cell imaging techniques rely on the use antibodies to label a specific target antigen with a fluorescent dye (also called fluorophores or fluorochromes) such as fluoresceinisothiocyanate (FITC). Primary conjugated antibodies that are chemically labeled to fluorophores are commonly used in IF.

What is the difference between conjugated and unconjugated antibodies?

The fluorophore allows visualization of the target distribution in the sample under a fluorescent microscope (eg epifluorescence and confocal microscopes). We distinguish between two IF methods depending on whether the fluorophore is conjugated to the primary or the secondary antibody:

Direct IF uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore.
Indirect IF uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.

The diagram below represents both direct and indirect methods.

Both methods have their advantages and disadvantages as shown in the table below.

	Direct	Indirect
Time	Protocols for direct IF are usually shorter as they only require one labeling step.	The fact that you have to use a conjugated secondary antibody to detect the primary antibody results in additional steps.
Cost	Conjugated primary antibodies are usually more expensive than their unconjugated counterparts.	Secondary antibodies are relatively inexpensive compared to primary antibodies. Further cost savings may be made by using the same conjugated secondary antibody to detect different primary antibodies.
Complexity	Fewer steps in the protocol simplify direct methods.	Added complexity in indirect methods may result from having to select the appropriate secondary antibody. This is particularly relevant in multiplex experiments where several secondary antibodies, each targeting a different species and conjugated to different dyes, are needed.
Flexibility	Commercially available pre-conjugated primary antibodies limit your flexibility.	The possibility of using different conjugated secondary antibodies adds greater flexibility.
Sensitivity	The signal obtained in direct methods may seem weak when compared to indirect methods as signal amplification provided by the use of secondary antibodies does not occur.	Several secondary antibodies will bind to the primary antibody resulting in an amplified signal.
Species cross-reactivity	Species cross-reactivity is minimized in direct methods as the fluorophore is already conjugated to the primary antibody.	Secondary antibodies may cross-react with species other than the target. The use of pre-adsorbed secondary antibodies can prevent cross-reactivity.
Background	Non-specific binding is reduced through the use of conjugated primary antibodies.	Samples with endogenous immunoglobulins may exhibit a high background with indirect methods.

Direct and indirect methods are not limited to immunofluorescence. They are also relevant to other techniques that rely on the use of fluorophore-conjugated antibodies such as flow cytometry, ELISA, western blot and immunohistochemistry.

Detection of low abundance proteins can be sometimes challenging even with indirect methods. Biotinylated antibodies offer an extra layer for increased signal amplification. Learn more about how methods based on the use of biotin-conjugated antibodies work here.