Indirect Labeling

Indirect immunofluorescence labeling is labeling in which the antibodies do not have the fluorescent dye attached. Indirect labeling is more involved than direct labeling. If you need to label cells with different antibodies for simultaneous analysis, you may encounter problems choosing secondary antibodies (fluorochrome conjugated antibodies that bind to the primary antibody). For example, if both primary antibodies are mouse IgG 1, what can you do to label them individually with different fluorochrome conjugated secondary antibodies? (FITC-conjugated anti-Mouse IgG 1 will label both!) The biotin-avidin system may provide an answer, if one antibody can be biotinylated. This is done by labeling with a non-biotinylated mouse Ab first followed by, the biotinylated mouse Ab and finally PE-streptavidin. Another example may be when one of the primary antibodies is available in a fluorochrome-conjugated form; incubate the cells with it after the primary and secondary antibody steps. This can obviously become very complicated so the best approach to concomitant labeling is to get all direct conjugates if possible and use the method for Direct Labeling.


  1. Primary Antibodies:
    • Test antibody: Usually unconjugated mouse monoclonal IgG or IgM. For labeling cells with more than one test antibody simultaneously, you need primary antibodies made in different animals or that have different Ig subclasses. Another option is to have one of them conjugated to biotin.
    • Negative control sera: Purified protein (usually mouse IgM or IgG of the same subclass) to match your test antibodies.
    • Secondary antibody: Fluorochrome-conjugated antibodies directed towards the protein composing the primary antibodies and negative control sera.
    • Fluorochrome-conjugated streptavidin: Use this if one of your primary antibodies is biotin conjugated.
  2. PBS with 0.1% sodium azide added. The sodium azide assists in preventing capping and shedding or internalization of the antibody-antigen complex after the antibodies bind to the receptors.
  3. Cells in suspension, counted and viability-checked. Keep them in culture medium supplemented with antibiotics and 2-5% fetal bovine serum, on ice. If viability is less than 90%, consider adding another fluorochrome to identify dead cells during analysis. 


  1. Adjust cell concentration to 1 million cells per ml.
  2. Place 1 ml of the cell suspension into a 12x75 tube. (One tube for each primary and secondary antibody combination plus the negative control).
  3. Centrifuge at 250 x g for 5 minutes. Using a pipet remove the liquid being careful not to disturb the pellet.
  4. Add the appropriate amount of primary antibody to your tubes. Proper Ab concentration should be determined in advance via titration, using known target cells with a large number of positive receptors.
  5. Vortex, and place cells on ice in covered bucket for 30 minutes.
  6. Wash cells by adding 1 ml of PBS w/ azide, vortexing, centrifuging and removing liquid as above.
  7. Repeat washing step for a second wash.
  8. Add the appropriate amount of secondary antibody or fluorochrome-conjugated streptavidin.
  9. Vortex, and place cells on ice in covered bucket for 30 minutes.
  10. Wash cells twice with PBS/azide as before and resuspend in 1ml PBS/azide. Vortex and place on ice till anyalysis.

Note: Lymphoid cells will generally last for several hours although that long a wait is generally not recommended. If you expect to be waiting that long or longer you should consider Fixing Your Cells which will preserve them for at least several days.