Encased against all protocols4/10/2023 Together, these four parts of an immunostaining protocol work in concert to create the ideal conditions for a highly specific binding of the primary antibody to a single protein, and proper visualization of this protein. The immunostaining process can be broken down into four major parts: 1) blocking and permeabilization (where required), 2) primary antibody addition, 3) secondary antibody selection and addition, and 4) detection. Immunostaining principles are very similar to those of a western blot but instead of purifying and immobilizing proteins onto a membrane, they are immobilized where they naturally cluster within the cell (i.e. The slightly acidic buffer citrate (pH 6.0) is effective at unmasking a wide range of epitopes, but some epitopes may require a more basic buffer, like EDTA (pH 8.0). The pH of the buffer helps keep the proteins unwound after the temperature has returned to normal, so the pH range of the system should be optimized to the antibody-epitope interaction of interest. HIER involves heating and then cooling the tissue sections while they are immersed in a solution with a defined buffering capacity. Either method can unmask epitopes, rendering them accessible to the primary antibody and amenable to staining by IHC.Īt CST, our most common antigen retrieval method is HIER, so this is the method we will discuss in detail. These include proteolytic-induced antigen retrieval, which relies on an enzyme like proteinase K, or heat-induced epitope retrieval (HIER), which uses heat to break apart cross-linked bonds and unwind proteins. Several methods exist for revealing epitopes that have been masked by fixation. When the tissue section is cut, it is mounted to the slide and allowed to air dry before the next steps. The type of blade chosen should match the instrument being used and the tissue being cut and it should be thoroughly cleaned between applications to maintain its sharpness. Despite many differences, one thing is common to all these sectioning instruments: the use of a very sharp blade to slice through tissue without leaving behind any damage. Once the tissue is stabilized for cutting, different instruments may be used to generate sections of various thicknesses, depending on the desired IHC application. Depending on the fixation method chosen, the type of block may vary from paraffin-based (for cutting using a microtome), to temperature-sensitive and water-soluble (for cutting with a cryostat), or agar (for cutting on a vibratome). EmbeddingĪfter fixation, the tissue needs to be stabilized in a block so that it can be cut into thin sections for staining. Chemical fixation is the most popular option but since other options exist, this initial step of should be considered carefully and the best fixation method should be determined based on how the rest of the IHC will be performed. Different chemicals can be used to lock the proteins of the cells and tissue into a rigid structure, creating a fixed skeleton of the previously alive cells. So the first, often overlooked, but very critical step of any IHC procedure, is fixation. Please Note: The following steps reflect the protocol for paraffin-embedded samples.Ī key feature of IHC is the preservation of tissue using one of the many available fixatives in order to maintain the native structure of the cells that make up that tissue. This guide aims to help you improve your IHC analysis by providing suggestions to allow you to achieve the expected results with minimal end-user optimization. IHC is a challenging application and problems often occur. We provide recommendations on reagents and procedures based upon our extensive experience with IHC as part of our antibody validation and technical support processes. Here we give an overview of our recommended protocol and discuss which steps we believe are key to a successful experiment. First, let’s go through a general outline of the important steps that are needed for an IHC experiment to be successful, then we’ll go through each step in more detail. (2012) Clin Cancer Res 18, 4449-4457.) How does immunohistochemistry work?Īs mentioned above, on its most basic level, IHC is dependent upon antibody detection of specific proteins of interest in a tissue sample. Note: Staining is of FIG-ROS1 fusion (Rimkunas, V.M. Immunohistochemical analysis of paraffin-embedded human lung carcinoma using ROS1 (D4D6) Rabbit mAb #3287.
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