The generalized procedure for immunofluorescence labeling of adherent cell cultures involves fixation of the cells with a suitable reagent, such as paraformaldehyde, glutaraldehyde, acetone, or methanol. The organic solvents serve double duty by also removing most of the plasma membrane components, thus exposing the interior of the cell to antibodies and other non-permeant reagents. Cell cultures fixed with aldehydes require a permeabilization step with a detergent. Triton X-100 is a popular permeabilizing agent, as are saponin, Tween, and sodium dodecyl sulfate (SDS). After permeabilization, cells are treated with a primary antibody (or a mixture of two different antibodies from different hosts) followed by a fluorophore conjugated secondary antibody. In some cases, the primary antibody is labeled with a fluorophore. After immunostaining, the cells can be counterstained with other reagents to visual structural details not revealed by the fluorescent antibodies.

In order to simultaneously visualize both the actin and tubulin networks in MDCK cells to capture the digital image illustrated above, a fixed and permeabilized culture was treated with mouse anti-alpha-tubulin primary antibodies, followed by a cocktail of goat anti-mouse secondary antibodies conjugated to Alexa Fluor 568 mixed together with Alexa Fluor 350 conjugated to phalloidin. Nuclei were counterstained with SYTOX Green. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.