Immunohistochemistry (IHC) Protocols

Immunohistochemistry

Immunohistochemistry (IHC) is a critical immunoassay for many laboratories and is used to demonstrate the presence or absence of important proteins, detect post-translational modifications, diagnose disease, and more. Rockland produces highly active antibodies and conjugates for use in IHC experiments. Rockland also produces high-quality reagents for setup and detection in IHC experiments. Protect your experiments with Rockland Antibodies.

IHC uses immunological and biochemical techniques to examine different tissue sections using labeled antibodies that interact with target antigens. A visible label or stain is used to visualize the interaction of the antibody and antigen. IHC enables the visualization of distinctive cellular components of cells and is frequently used by pathologists and diagnostics.

IHC sample preparation

The antigen is fixed and adhered to a glass microscope slide. Formaldehyde fixation is suggested as the initial method of routine choice for tissue and cell fixation, but other methods are also suitable. Background staining is reduced with a blocking buffer to block non-specific sites to which antibodies can bind. Rockland offers common blocking buffers including BSA, normal whey, skimmed milk powder, and a proprietary optimized blocking buffer.

IHC sample labeling

Labeling is accomplished by reacting a primary antibody with immobilized antigen to form an antigen-antibody complex. A second biotinylated antibody specific for the host species of the primary antibody reacts with the complex. Streptavidin conjugated to the enzyme peroxidase or alkaline phosphatase reacts with this complex. Finally, a substrate is added that causes a reaction with the enzyme, producing a colored precipitate that forms on the slide at the location of the antigen. The slide is viewed through a standard light microscope and photographed. A common manipulation in IHC binding is the use of the antigen retrieval technique, a simple method of heating or boiling paraffin-embedded tissue sections to enhance the observed signal.

Immunohistochemistry Kits

MaxTag ™ Histoimmunohistochemistry Kits allow detection of user-supplied primary polyclonal or monoclonal antibodies. The kits are based on the high specificity of avidin-biotin binding for the visualization of an antigen and are intended to provide a simple, reliable and convenient detection system for immunohistochemical staining. Kits include all necessary reagents for IHC, such as an antibody, buffers and substrates, fixative, and counterstain. These easy-to-use kits guide you through IHC using highly optimized procedures that result in low background and high signal, producing the most sensitive detection kit available.

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