Immunohistochemistry (IHC) is an antibody-based laboratory technique that allows the user to detect a specific protein within a section of tissue taken from a plant or animal. Labelled antibodies alongside counterstains help localise the protein of interest within the tissue section, giving a wealth of information useful not only to researchers, but to histopathologists who use IHC as a diagnostic tool.
Many options are available when devising an IHC experiment. Tissues can be snap-frozen or embedded in paraffin before sectioning under a cryostat for frozen sections, or a microtome for paraffin embedded sections. There are also various tissue fixation methods and a multitude of antibody labels for detection of antigen. Frequently used labels include horseradish peroxidase (HRP), alkaline phosphatase (AP) and biotin for chromogenic detection or fluorochrome-labelled antibodies for fluorescent detection.
Below are some basic considerations to take into account when designing or troubleshooting an immunohistochemistry protocol:
Preparation of sections
In order to preserve the tissue and antigen under investigation, various fixation and preservation techniques are employed. Common tissue fixation methods include 10% neutral-buffered formalin (NBF), 100% acetone or alternatively methanol, and 4% formaldehyde. Use of an incompatible fixation method is a frequent cause of poor antigen detection in IHC protocols. Certain epitopes are especially sensitive to the type of fixation method, and fixation time is crucial, as over-fixation can lead to masking of the antigen. Usually, the antibody datasheet will provide guidance on the best fixation method, otherwise acetone-fixed frozen sections, or formalin-fixed paraffin-embedded sections (FFPE) are considered standard and a good starting-point.
Snap-freezing of sections in liquid nitrogen is a reliable method favoured when the preservation of enzyme function and antigenicity is important, or if an epitope is phosphorylated. Sections treated this way require a simpler staining protocol compared to paraffin-embedded sections, and as they are often fixed with alcohol, they do not have issues with antigen masking. Though, in comparison to paraffin embedded sections they have a reduced shelf life, and require cold storage at -80°C.
Paraffin embedding of sections preserves tissue morphology and allows the more convenient storage of sections at room temperature, with an extended shelf life of multiple years. However, the sections require de-paraffinization in solvent before they can be stained, and formalin-fixed (FFPE) sections may suffer from antigen masking requiring an antigen retrieval procedure.
Antigen retrieval is required where fixation has caused cross-linking, thus restricting access to the antigen of interest. A selection of methods are available to achieve epitope unmasking; proteolytic-induced epitope retrieval (PIER) uses proteases such as trypsin, proteinase K or pronase; heat-induced epitope retrieval (HIER) unmasks antigen using a combination of heat and buffer. Commonly used HIER buffers are citrate buffer, EDTA buffer and Tris/EDTA buffer. Again, there may be guidance provided on the antibody datasheet as to whether antigen retrieval is necessary, and if a specific method is recommended. If not, a variety of retrieval techniques may have to be tested for their effectiveness. In certain cases, the duration of formalin fixation can affect which retrieval method gives the best results.
As with most antibody-based protocols, appropriate blocking for non-specific antibody staining is required. If using an indirect staining method, this is usually 10% normal serum from the same species in which the secondary antibody was raised. Additional blocking for IHC is advised to limit background staining caused by endogenous biotin when using biotin-based detection, and endogenous peroxidases or phosphatases prior to using horseradish peroxidase (HRP) or alkaline phosphatase (AP) antibody conjugates. For ease of use, a variety of blocking buffers are commercially available.
Not all antibodies are suitable for use in immunohistochemistry, information such as tested applications (IHC on frozen or paraffin-embedded sections), working dilutions, fixation and antigen retrieval methods are an advantage when selecting an antibody for IHC. Antibody staining can be direct or indirect. Direct staining requires a conjugated primary antibody and is ideal when the antigen has high expression levels in the tissue. Indirect staining requires a conjugated secondary antibody and allows amplification of signal for harder to detect epitopes. If using an indirect protocol, include a secondary antibody only control (no primary antibody), to determine if the secondary antibody is binding non-specifically to the tissue section. Also, the primary antibody should be raised in a different species to that of the tissue sample.
Quality of sections
Many IHC experiments fail due to the quality of the section itself. Never allow slides to dry out during the immunostaining procedure. Store slides as recommended and note that slides can deteriorate over time.
Any reagent used in an assay must be within its expiry date and stored as recommended by the manufacturer. Follow the manufacturer’s instructions for use, but be aware that optimisation of experimental conditions, such as incubation times and antibody concentrations, may be required for the best results in your laboratory setting.
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