News & Blog: Article

Getting more out of your mouse monoclonal antibodies: multiplex staining with subclass specific secondaries.

Contributed by our own Kristen Kralovich who has spent many an hour behind the objectives of a microscope grabbing brilliant images for papers and datasheets alike.

Multiplex immunofluorescent staining allows for simultaneous detection of multiple protein targets in a single sample, tissue or cell at the same time by using differently colored fluorescent labeled secondary antibodies. There are many benefits of multiplex staining including:

-the ability to acquire spatial and colocalization information
-an increase in the amount of data acquired from each sample and in experiments
-the ability to study several targets at once, saving valuable samples and reagents

Multiplex staining protocols have been developed for many assays including western blotting, IF, IHC, flow cytometry and ELISA (FLISA). An increasing number of fluorophores are also being developed allowing for a larger number of targets which can be visualized in one assay.

Although multiplex labeling is common, some researchers may feel limited to using primary antibodies from different species to accomplish this staining. Sometimes, quality antibodies against the the desired target(s) are not available in a second or third host species. This issue can be resolved by using mouse subclass specific secondary antibodies. More than one mouse monoclonal antibody can be used together to perform multiplex staining of a single sample. Additionally, staining with subclass specific antibodies provides cleaner staining with less background signal and less “false positive” staining.

Monoclonal antibodies developed for research use are typically developed from a few highly tested mouse strains which may include Balb/c, C57Bl/6, C57Bl/10, and CD1. Mice produce immunoglobin classes (isotypes) IgA, IgD, IgE, IgG and IgM where each class has a different sequence in the constant region of the antibody heavy chain. Research applications rely heavily on the IgG class of antibodies which is subdivided into subclasses IgG1, IgG2 and IgG3 antibodies. Finally, the IgG2 subclass is further subdivided into subclasses IgG2a, IgG2b and IgG2c. The IgG class is the most abundant class in serum (although other classes do have research applications and may be used as well).

The isotype and subclass of a monoclonal antibody should be indicated on the manufacturer’s datasheet. Using each primary monoclonal antibody with a secondary antibody that is specific for the isotype and subclass of the primary antibody allows more than one mouse antibody to be used in a single sample, providing the antibodies are of different subclasses.

Antibodies Incorporated offers a large collection of mouse monoclonal antibodies spanning the different IgG subclasses. In many cases we offer two antibodies against the same protein target from different subclasses, increasing your multiplexing possibilities. For example, we offer 3 different subclasses of mouse monoclonal α-GFP: IgG1 (N86/20), IgG2a (N86/8 and N86/38) and IgG2b (N86/44). As an example, multiplex staining for both tyrosine hydroxylase (TH) and GFP could be done with our highly cited mouse monoclonal α-TH antibody LNC1 (subclass IgG1) in tandem with our mouse monoclonal α-GFP antibody N86/8 (subclass IgG2a). A number of our other popular antibodies are available in multiple subclasses to accommodate your multiplexing assays. These include: c-FOS, PSD-95, Ankyrin G and more!

Adult rat hippocampus CA1 immunofluorescence of axon initial segments multiplex-stained with the following mouse monoclonal antibodies: anti-Anykyrin-G N106/43 (IgG1) green, anti-Kv2.1 K39/25 (IgG2a) red, and anti-Kv1.2 (IgG2b) K14/16 blue.  Image courtesy of Matt Rasband (Baylor College of Medicine).