As with most fluorescence techniques, a significant problem with<unk>  is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing or limiting the intensity or time-span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g., Alexa Fluors, Seta Fluors, or DyLight Fluors). Some problems that may arise from this technique include autofluorescence, extraneous undesired specific fluorescence, and nonspecific fluorescence. Autofluorescence includes fluorescence emitted from the sample tissue or cell itself. Extraneous undesired specific fluorescence occurs when a targeted antigen is impure and contains antigenic contaminants. Nonspecific fluorescence involves the loss of a probe's specificity due to fluorophore, from improper fixation, or from a dried out specimen.<unk>  is only limited to fixed (i.e., dead) cells when structures within the cell are to be visualized because antibodies do not penetrate the cell membrane when reacting with fluorescent labels. Antigenic material must be fixed firmly on the site of its natural localization inside the cell. Intact antibodies can also be too large to dye cancer cells in vivo. Their size results in slow tumor penetration and long circulating half-life. Research has been done investigating the use of diabodies to get around this limitation. Proteins in the supernatant or on the outside of the cell membrane can be bound by the antibodies; this allows for living cells to be stained. Depending on the fixative that is being used, proteins of interest might become cross-linked and this could result in either false positive or false negative signals due to non-specific binding. An alternative approach is using recombinant proteins containing fluorescent protein domains, e.g., green fluorescent protein (GFP). Use of such "tagged" proteins allows determination of their localization in live cells. Even though this seems to be an elegant alternative to<unk> , the cells have to be transfected or transduced with the GFP-tag, and as a consequence they become at least S1 or above organisms that require stricter security standards in a laboratory. This technique involves altering the genetic information of cells.
Immunofluorescence
Immunostaining
Immunohistochemistry
Immunolabeling
Gluten immunochemistry
Immunopathology
Direct fluorescent antibody
Reticulin stain