histological section of human skin prepared for direct<unk>  using an anti-IgG antibody. The skin is from a patient with systemic lupus erythematosus and shows IgG deposit at two different places: The first is a band-like deposit along the epidermal basement membrane ("lupus band test" is positive). The second is within the nuclei of the epidermal cells (anti-nuclear antibodies). Preparation of fluorescence To make fluorochrome-labeled antibodies, a fluorochrome must be conjugated ("tagged") to the antibody. Likewise, an antigen can also be conjugated to the antibody with a fluorescent probe in a technique called fluorescent antigen technique. Staining procedures can apply to both fixed antigen in the cytoplasm or to cell surface antigens on living cells, called "membrane<unk> ". It is also possible to label the complement of the antibody-antigen complex with a fluorescent probe. In addition to the element to which fluorescence probes are attached, there are two general classes of<unk>  techniques: primary and secondary. The following descriptions will focus primarily on these classes in terms of conjugated antibodies. There are two classes of<unk>  techniques, primary (or direct) and secondary (or indirect). Primary (direct) Primary (direct)<unk>  uses a single, primary antibody, chemically linked to a fluorophore. The primary antibody recognizes the target molecule (antigen) and binds to a specific region called the epitope. This is accomplished by a process which manipulates the immune response of organism with adaptive immunity. The attached fluorophore can be detected via fluorescent microscopy, which, depending on the messenger used, will emit a specific wavelength of light once excited. Direct<unk> , although somewhat less common, has notable advantages over the secondary (indirect) procedure. The direct attachment of the messenger to the antibody reduces the number of steps in the procedure, saving time and reducing non-specific background signal. This also limits the possibility of antibody cross-reactivity and possible mistakes throughout the process. However, some disadvantages do exist in this method. Since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct<unk>  is substantially less sensitive than indirect<unk>  and may result in false negatives. Direct<unk>  also requires the use of much more primary antibody, which is extremely expensive, sometimes running up to $400.00/mL. Secondary (indirect) A fluorescent stain for actin in the smooth muscle of the skin. Secondary (indirect)<unk>  uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognizes the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time-consuming than the primary (or direct) protocol above, but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody. This protocol is possible because an antibody consists of two parts, a variable region (which recognizes the antigen) and constant region (which makes up the structure of the antibody molecule). It is important to realize that this division is artificial and in reality the antibody molecule is four polypeptide chains: two heavy chains and two light chains. A researcher can generate several primary antibodies that recognize various antigens (have different variable regions), but all share the same constant region. All these antibodies may therefore be recognized by a single secondary antibody. This saves the cost of modifying the primary antibodies to directly carry a fluorophore. Different primary antibodies with different constant regions are typically generated by raising the antibody in different species. For example, a researcher might create primary antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit secondary antibodies that recognize the goat antibody constant region ("rabbit anti-goat" antibodies). The researcher may then create a second set of primary antibodies in a mouse that could be recognized by a separate "donkey anti-mouse" secondary antibody. This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.
Immunofluorescence
Immunostaining
Immunohistochemistry
Immunolabeling
Gluten immunochemistry
Immunopathology
Direct fluorescent antibody
Reticulin stain