Immunohistochemical Methods

The Enzymatic method for immunohistochemistry uses reagents like Calcium Chloride, Sodium Hydroxide, Hydrochloric Acid solutions, Xylenes for dewaxing, and Methanol. Immunohistochemistry use different staining procedures such as one step direct method, ABC methods, two-step indirect method and Tyramide signal amplification.

Direct Method:

Direct method is one step staining method, and involves a labeled antibody (i.e. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method.

Indirect Method:

Indirect method involves an unlabeled primary antibody (first layer) which react with tissue antigen, and a labeled secondary antibody (second layer) react with primary antibody (Note: The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised). This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. In addition, it is also economy since one labeled second layer antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens.

The second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine or Texas red, and this is called indirect immunofluorescence method. The second layer antibody may be labeled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase, and this is called indirect immunoenzyme method.

PAP Method (peroxidase anti-peroxidase method):

PAP method is a further development of the indirect technique and it involves a third layer which is a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase anti-peroxidase complex. The complex, composed of rabbit gaba-globulin and peroxidase, acts as a third layer antigen and becomes bound to the unconjugated goat anti-rabbit gaba-globulin of the second layer. The sensitivity is about 100 to 1000 times higher since the peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound, and loses none of its enzyme activity. It also allows for much higher dilutionof the primary antibody, thus eliminating many of the unwanted antibodies and reducing non-specific background staining.

Avidin-Biotin Complex (ABC) Method:

ABC method is standard IHC method and one of widely used technique for immunhistochemical staining. Avidin, a large glycoprotein, can be labeled with peroxidase or fluorescein and has a very high affinity for biotin. Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies.

The technique involves three layers. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin peroxidase. The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products.

Labeled StreptAvidin Biotin (LSAB) Method:

Streptavidin, derived from streptococcus avidini, is a recent innovation for substitution of avidin. The streptavidin molecule is uncharged relative to animal tissue, unlike avidin which has an isoelectric point of 10, and therefore electrostatic binding to tissue is eliminated. In addition, streptavidin does not contain carbohydrate groups which might bind to tissue lectins, resulting in some background staining.

LSAB is technically similar to standard ABC method. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is Enzyme-Streptavidin conjugates (HRP-Streptavidin or AP-Streptavidin) to replace the complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solutions to produce different colorimetric end products. The third layer can also be Fluorescent dye-Streptavidin such as FITC-Streptavidin if fluorescence labeling is preferred.

A recent report suggests that LSAB method is about 5 to 10 times more sensitive than standard ABC method.