Definition The demonstration of antigen in tissue section or smears by the use of specific antigen/ antibody, which culminate in the attachment of a marker to the antigen. For light microscopy, the marker may be a fluorescent dye, an enzyme or colloidal gold. Immunosytochemistry is now recognized as an element and a major tool; both in diagnostic and research orientated cellular pathology. The technique involves the detection of specific or highly selective cellular epitopes with an antibody and appropriate labeling system. With many antibodies identifying epitopes that survive formaline fixation and processing to paraffin wax and clinical data alone cannot provide a diagnosis.There are numerous immunocytochemical techniques, which may be used to localize antigens. The selection of a suitable technique should be based on parameters such as the type of specimen under investigation and the degree of sensitivity required. Cost may also be a factor.  Direct Method In this technique a labeled antibody reacts directly with the antigen in the histological or cytological preparation. The technique however provides little signal amplification and is rarely used on paraffin sections. The main application remains in immunofluorescence, where this technique is used to identify immunoglobuline and complement in frozen section of skin and renal biopsies. Direct (Enhanced Polymer On-Step Technique) This method uses a dextran polymer backbone to which are conjugated large number of primary antibody molecules and peroxidase enzyme. This produces a sensitivity similar to that achieved with an avidin-biotin system but with fewer steps. The reagent are supplied optimally diluted for 1 hour incubation and the advantage of this technique are that it is rapid, especially for frozen section immunocytochemistry, and sensitive enough to detect small amounts of antigen. The main disadvantage with EPOS is that the numbers of antibodies supplied in this form are limited and, as with prediluted reagent, the scope for optimizing the system in-house is very limited. Indirect An unconjugated primary antibody react with the antigen and enzyme-labeled secondary antibody is directed against the primary. This technique is more versatile as a variety of primary antibodies from the same species can used with the same-labeled secondary antibody. This is also more sensitive than the direct technique as several secondary antibodies can react with different epitopes on the primary antibody, which leads to more enzyme deposition at the site of the antigen. Indirect (EnVision+) The technology used here is similar to that used in the EPOS system, employing a dextran polymer backbone that has a large number of antibody molecules and enzyme molecules attached. The advantage of this technique over EPOS is that a number of different primary antibodies from the same species can be used, with the same labeled secondary layer. A big advantage of both EPOS and EnVision+ is that they can be used on tissue that contains a lot of endogenous biotin without producing background staining. Peroxidase-anti peroxidase This used a peroxidase-anti peroxidase (PAP) complex that consists of 3 molecules of peroxidase and 2 antibody molecules directed against peroxidase. This technique is useful for tissues that contain a lot of endogenous biotin. .e.g. renal and liver biopsies. Alkaline phosphatase-anti alkaline phosphatase This uses an alkaline phosphatase-anti alkaline phosphatase (APAAP) complex, which consists of 2 molecules of alkaline phosphatase and 1 molecules of antibody, directed against alkaline phosphatase. This technique is useful for tissues that contain a lot of endogenous peroxidase, e.g. blood smears and cytology preparation. Avidin-Biotin Technique These methods utilize the high affinity of the glycoprotein avidin for biotin, a low molecular weight vitamin. Avidin –biotin techniques provide high sensitivity as the high affinity of streptavidin for biotin enables firstly a very stable complex to be formed and secondary, many enzyme molecules to be deposited at the antigenic site. Enzyme Histochemistry Enzymes are proteins synthesized in the living cell where they act as biological catalysts. The substance or chemical acted upon by an enzyme is known as its substrate and since enzymes are highly specific they tend to be limited to one substrate. In this demonstration of an enzyme therefore depends upon their effect on a given substrate. In this way their visualization differs from that of inactive tissue component since enzyme must be active to be demonstrated. One cannot see an enzyme only the effect it has upon a substrate therefore the basis of all enzyme histochemical reactions relies upon the formation of an insoluble substance at the site of activity of an enzyme on a specific substrate. This insoluble substance has then to be rendered colored if not already visible. And the most tissues in used in our lab.is the Muscle biopsy. The main enzyme histochemistry staining used in our laboratory are: Alkaline phosphates. Acid phosphates Dehydrogenases.( NAD/NADH / Succinate / G6Po4). Specific Phosphastase ( ATPase).in different PHs.