AAT Bioquest

What are the two types of immunofluorescence?

Posted June 24, 2022


The two main types of immunofluorescence assays are primary and secondary.  

Primary or Direct immunofluorescence 

Primary immunofluorescence involves chemical linking a single primary antibody to a fluorophore. The primary antibody recognizes and binds to a specific region called the epitope of the target molecule. The direct attachment of the antibody to the fluorophore offers several advantages. There are fewer steps involved, which makes the process faster. It also minimizes background signal and reduces antibody cross-reactivity. The downside of primary immunofluorescence is that only a limited number of fluorescent molecules can bind to the primary antibody. This reduces its sensitivity and increases the incidence of false negatives. 

Secondary or Indirect Immunofluorescence

Secondary immunofluorescence involves the use of two antibodies – an unlabeled primary antibody and a secondary antibody that carries the fluorophore. The unlabeled primary antibody binds the target molecule. The secondary, fluorophore-carrying antibody recognizes the primary antibody and binds to it. A single primary antibody can be bound by multiple secondary antibodies. This provides higher signal amplification because there are more fluorophore molecules per antigen. This process is more flexible because a variety of secondary antibodies as well as detection techniques can be used for a single type of primary antibody. The downside of this process is that it takes more time because there are more steps involved. 

Additional resources

An Introduction to Performing Immunofluorescence Staining

ReadiLink™ BSA Conjugation Kit

Enzyme-Linked Immunosorbent Assay (ELISA)