Why: To identify a specific organelle or another cellular structure and to mark individual cells, it is necessary to counterstain them in immunocytochemistry/immunofluorescence (ICC/IF) assays.
How: Counterstaining is often performed with dyes or antibodies specific to the organelle or cellular structure of interest. For example, the nuclear counterstaining is carried out by using DNA helix intercalating dyes such as DAPI and Hoechst which can penetrate the cells and nuclei without permeabilization. Similarly, fluorescently-labeled phalloidin is used for counterstaining the cytoskeletal actin filaments and fluorescently-tagged wheat germ agglutinin (WGA) is employed for counterstaining the plasma membranes.
Important Considerations: When choosing counterstaining options in ICC/IF, it is important to ensure that the chosen counterstain and fluorochromes in use for the target proteins are displaying a negligible spectral overlap. This will allow for an easy distinguishing of signals and in eliminating the bleedthrough effect. For example, DAPI (the most commonly used counterstain) is not used in combination with Alexa Fluor® 405, DyLightTM 405, or HiLyghtTM 405 all of which are blue color emitting fluorochromes. See an image below for the counterstaining of nuclei and actin filaments in ICC/IF analysis of DDX21, a nucleolar protein.
ICC/IF detection of DDX21 in HeLa cells which were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton X-100. The cells were incubated with anti-DDX21 [NB100-1718] at a 1:200 dilution overnight at 4°C and detected with an anti-rabbit DylightTM 488 (green) at a 1:500 dilution. Alpha tubulin (DM1A) [NB100-690] was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse DylightTM 550 (red) at a 1:500 dilution. Nuclei were counterstained with DAPI (blue) [NBP2-31156].
Review Organelle Marker Brochure for a complete list of organelle marker antibodies. Also, visit Counterstaining and Mounting in ICC/IF page for detailed information on various counterstaining dyes which are available from Novus Biologicals.
