Immunohistochemistry - getting the most out of your IHC protocol
The application of immunohistochemistry provides a visual opportunity to interpret tissue integrity, and allows for monitoring of the way in which protein expression is distributed across them. In previous years the application mainly visualised proteins using the chromogenic method, but more recently fluorescence IHC has started becoming a prominent competitor as researchers seek to visualise several proteins simultaneously in a single tissue sample.
Common as it is in both clinical and academic environments, immunohistochemistry is not the easiest of applications to get right, despite being a relatively straight forward procedure. The application commands a good level of technical skill throughout, and skilled researchers do well to acquire the appropriate resources for their expression system before getting started.
For those of you taking your first experience with immunohistochemistry we have prepared a series of tips to get started, or if you’re looking to brush up we hope the points made here will help you on your way to achieving quality IHC research data.
1. How should I prepare my tissue samples?
Two commonly applied tissue fixation techniques include formalin and freezing. Both have certain pitfalls, as freezing can reduce morphological integrity, and formalin fixing can cause cells to shrink – leading to higher antigen density. But in general, these processes help prevent degradation of your tissue and enhance the refractive index for visualising.
Most laboratories will have a technician skilled in sectioning tissues, however if you are looking to perform this stage solo, ensure that your sections are smooth and uniform. Ensure that a cryostat or microtome is used for frozen sections or FFPE sections, respectively.
2. How to fix tissue for immunohistochemistry?
Whichever way you choose to fix your tissue, take caution to assess the suitability of the pH, duration of fixing and use of specific acids in the process. The application may require different conditions according to tissue type, age and the antigen you seek to visualise. As there is no definitive answer to this, we would recommend using the literature to research the most suitable conditions.
|Formalin Fixed||Frozen Sections|
|Advantages||Preferred for phosphorylated proteins|
|Disadvantages||Cell shrinkage||Reduce morphological integrity|
3. Antigen retrieval – which induction system should I use?Fixing your tissues can mask the antigen you need access to through protein cross-linking. If the primary antibody can’t bind to it, the application will fail. We’ve made a table for 2 common methods of retrieval. Both have distinct advantages and disadvantages, although the heat-induced method is more widely used due to the potential morphological changes which can arise from enzyme-induced antigen retrieval.
|Incubation period||5-30 minutes||10-20 minutes|
|Buffer||Neutral buffer solutions of enzymes such as pepsin, proteinase K or trypsin||Depends on pH required. Sodium citrate pH6.0, EDTA pH8.0 or Tris-EDTA pH10.0|
|Conditions pH||pH 7.4||Citrate buffer of pH6.0 or above. We recommend optimising for best results.|
|Precautions||Risk of enzyme reaction altering morphology of the tissue or antigen integrity.||Boiling can remove tissue section from slideMicrowaving can result in uneven heat distribution.|
4. Blocking the endogenous enzymes – What impact do these have?Biotin presents endogenously in tissues and can have a profound impact when it comes to visualising those tissues. A misinterpreted false positive would be costly to your overall research. Avidin-Biotin blocking kits are important to prevent this. Peroxidases and phosphatase enzymes are also found endogenously. Use a suitable inhibition process to prevent their activity and the development of unwanted artefacts.
5. How to keep the background signal low?...Block non-specific binding sitesIf you know the term, too many cooks spoil the broth, the same can be said for too many unnecessary binding sites. Non-specific binding sites, unsurprisingly, can give rise to unwanted non-specific signals. Inhibiting these will help keep your tissue background low, improving accuracy and precision in your work. Block non-specific binding sites using serum from the same species as your secondary antibody. If this isn’t available, universal blocking agents are also available on the market.
6. Have I got the right detection system for Immunohistochemistry?The greater the volume density of your target protein, the more suitable it will be for using a conjugated primary antibody, to give a well-defined signal. However, if antigen expression is not so high, using a secondary antibody can render a strong signal, because the signal may be amplified. Highly expressed antigens – choose a conjugated primary Low-to-medium antigen expression – use a conjugated secondary antibody because the signal can be amplified. Avidin-biotin – most commonly used detection kits. Biotin is very difficult to fully block, and non-specific staining may still occur. Alternatives are also available which use enzyme-conjugated polymers and do not require as high volumes of primary antibody to use.
7. Which chromogen or fluorochrome should I use for IHC?Choosing a suitable colour is important, and not because it looks pretty. Enzymatic-substrate reactions may take time to produce colour or impact other organic compounds used in the procedure. A fast reaction would be ideal which only takes place between the conjugate and desired substrate and does not interfere with other organic molecules found in media and counterstains. You’ll also want to consider the intensity of the counterstains, and how these will contrast or impact the mounting media. DAB for example is the substrate of choice for an HRP detection kit. The reaction is fast, precise and is compatible with common organic media and counterstains.
8. Counterstain & mount - How does your counterstain contrast with your chosen chromogen?There are many counterstains which are used in IHC. Below is a table of some of the more common ones and their features.
|Haematoxylin||Nuclear||Blue-purple||Binds to Lysine residues on nuclear histones, creating a metallic aluminium metal-dye complex. Takes up to 1 hour.|
|Fast red / Kernechtrot dye||Nuclear||Red||Fast reacting, within 5 minutes.|