Five key tips for a better multicolor immunofluorescence staining

1. Multicolor immunofluorescence staining is best carried out by sequentially incubating cells with unlabeled-primary and labeled-secondary antibodies. However when options are limited, it may also be performed by simultaneous incubation of cells with directly labelled primary antibodies.  
   
2. When picking fluorochrome colors for labeled primary or secondary antibodies, one must consider their fluorescent emission spectrum. Ensure that there is a minimal to negligible emission-spectra overlap between the selected fluorochromes. This will help eliminate bleed through effects during staining analysis.  
   
   
3. It is recommended to use the dimmest fluorochrome from one's available options to label the most abundant protein. Conversely, one should try detecting the least abundant protein with the fluorochrome having the highest quantum yield. These tips will help to efficiently/accurately detect multiple proteins in immunofluorescence staining.  
   
   

   MAP2 Antibody (5H11) [NBP1-92711] Mixed neuron/glia cultures from rat stained with MAP2 antibody (5H11) [NBP1-92711] (green) and NF-H antibody [NB300-135] (red). Since the NF-H protein is largely expressed in neuronal axons and MAP2 is only found in neuronal dendrites and perikarya, there is little overlap between these two staining patterns.

   
   
4. For reducing fixative-induced auto-fluorescence, try quenching the fixation agent to eliminate any free aldehyde groups, which could non-specifically bind the antibody. Quenching of formaldehyde, the most commonly used fixative, is performed with 0.1M Tris or Glycine buffer. Glutaraldehyde can be quenched with 0.1% sodium borohydride.  
   
   
5. During the entire staining procedure, treat the cells on coverslips very gently. Keep the mechanical manipulations to a minimum to avoid artifacts in staining. Never let cells dry out and avoid dropping solutions directly on them. This will help maintain the proper cellular structural morphology and localization of proteins.

Also see: Multicolor Immunocytochemistry/ Immunofluorescence (ICC/IF) staining protocol

Compiled by: Subhash Gangar