酶免疫组织化学技术英文

酶免疫组织化学技术英文

Enzyme immunohistochemistry (EIH) is a widely used technique to localize and visualize specific molecules in tissue sections. This technique involves the use of enzymatic reactions to generate colorimetric or fluorescent signals

that can be detected by microscopy. In this article, we will walk through the steps involved in EIH and its applications

in research.

Step 1: Sample Preparation

Before undertaking EIH, the tissue samples need to be appropriately prepared. This involves the fixation of the tissue in a suitable fixative, such as formalin, followed by embedding in paraffin. After this, the tissue blocks are

sliced into thin sections (3-5 μm thick) and mounted onto glass slides.

Step 2: Antigen Retrieval

Antigen retrieval is a crucial step in EIH where the epitope

of the target protein is exposed to the antibody. This is achieved by using heat or enzymatic treatment. Heat-induced antigen retrieval (HIAR) is commonly used, where the tissue slides are heated in a retrieval buffer, such as citrate or Tris-EDTA, in a pressure cooker or microwave oven. Alternatively, proteolytic enzymes, such as pepsin or trypsin, can be used to digest the tissue sections.

Step 3: Blocking of Non-specific Binding

To avoid non-specific binding of the primary antibody to the tissue, non-specific binding sites on the slide are blocked. This can be done by incubating the tissue sections with a

blocking agent, such as serum or bovine serum albumin.

Step 4: Primary Antibody Incubation

In this step, the tissue sections are incubated with the primary antibody that recognizes the target antigen. The antibody can be either monoclonal or polyclonal and can be supplied as a conjugate, such as peroxidase or alkaline phosphatase. The antibody is incubated with the tissue sections for a specific time at a suitable temperature.

Step 5: Secondary Antibody Incubation

After washing the tissue sections to remove any unbound primary antibody, a secondary antibody is added. The secondary antibody is conjugated to an enzyme, such as horseradish peroxidase, which will amplify the signal generated by the primary antibody.

Step 6: Enzyme Substrate Incubation

After washing the tissue sections again to remove any unbound secondary antibody, an enzyme substrate is added to the sections. The substrate will react with the enzyme, generating a visible signal. The most commonly used substrates for EIH include diaminobenzidine (DAB) and

alkaline phosphatase substrate (AP).

Step 7: Counterstaining and Mounting

To visualize the tissue structure, a counterstain can be added after the signal has been generated. Hematoxylin is commonly used as a counterstain to highlight the nuclei of cells. After counterstaining, the tissue sections are dehydrated and mounted under a coverslip.

Applications of EIH

EIH has numerous applications in research, including the detection of specific antigens in pathological tissues, the identification of cell types, and the evaluation of changes