Applications Focus: Labeling with multiple secondary antibodies

Multiple fluorescent labeling with secondary antibodies for immunocytochemistry and immunohistochemistry is a powerful tool to examine the behavior and interactions of more than one protein in a cell or tissue sample.  However, there are a few guidelines to follow to make sure your samples are correctly labeled. Read our top five tips for a successful multiple antibody labeling experiment:

1. Select primary antibodies raised in different host species. If you must use antibodies from the same host, use different IgG isotypes. Perform single staining of the antibodies prior to introducing multiple antibodies and secondary antibodies to the experiment, this will allow you to understand their isolated behavior and expression.
   
   
2. When selecting secondary antibodies, make sure they come from the same host species, but have different fluorophores within a safe range of spectra to avoid overlap. It is a good practice to use a stronger fluorophore for proteins with low expression and a weaker fluorophore for proteins with high expression to optimize visibility.
   
   
3. If you are using a primary antibody pre-conjugated to a fluorophore, you will simply add the conjugated antibody to your antibody blocking solution and rinse afterwards. If you are using a two-part detection method, you will first incubate cell or tissue samples with the primary antibodies of choice, rinse, and then introduce the secondary antibodies of choice. Primary antibodies that are pre-conjugated to secondary fluorophores can differ in their specificity, so proper testing is encouraged utilizing both methods to compare.
   
   

   +Primary/+Secondary               -Primary/+Secondary
   
   ICC/IF analysis of NFATc1 in MCF7 cells using NFATc1 antibody (7A6) [NB300-620] with DyLightTM 488 conjugated secondary antibody (green). Phalloidin (red) and DAPI (blue) were used for counterstaining the cytoskeleton and nuclei, respectively. The image on the right shows a control with all conditions being the same but without addition of primary antibody.

4. Be sure to stain your tissue or cell samples with the secondary antibodies alone, to assess background noise and potential bleed through before you introduce the primary antibody for binding. This step can help to troubleshoot secondary antibody specific needs. 
   
   
5. Become familiar with your fluorescent microscope and how to properly visualize double or triple stain fluorophores. Be cognizant of overexposure and photobleaching during image collection. 

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Brouns I. et al. (2002). Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors. J Histochem Cytochem. 50(4):575-82. DOI:10.1177/002215540205000415