Anyone who has perused a flow cytometry reagent website or catalog has noticed that hundreds of antibodies are commercially available. If you happen to be staining samples from humans, mice, or primates, odds are good that you can buy an existing fluorescently conjugated antibody for your specific target. But what do you do if you are looking at a novel cellular target or working with an animal species with fewer reagents available like dogs, guinea pigs, or fish.
Consider these three strategies for staining novel targets or species without the convenience of existing antibodies.
1. Use A Secondary Antibody
You may have an existing antibody to your target of interest, but it is not conjugated with a fluorochrome. Secondary antibodies that are conjugated with fluorochromes can bind to constant regions of primary antibodies and be detected on a flow cytometer. This approach is the most direct way to deal with an unconjugated primary antibody, but titrations must be done for both primary and secondary antibodies.
2. Test For Cross-Reactive Antibodies
Antibodies specific to highly conserved targets may be cross reactive between species, and human antibodies can often be used on primate samples. Check out your options for potentially cross-reactive antibodies as a work around.
3. Create Your Own Antibody
If neither of these options is feasible or successful, you can create your own antibodies using techniques in mice or rabbits to isolate monoclonal or polyclonal antibodies. This approach is costly and time-consuming, and often better done by a contract research organization familiar with antibody production. If you do isolate a good antibody, it can be conjugated with a fluorochrome or used with a secondary antibody.
These tips should help you keep your research on track and potentially create a new reagent that can benefit many scientists.