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.

	Enzyme-induced	Heat-induced
Temp	~37C	~95C
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 sites

If 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.

Name	Targets	Colour	Additional
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.
Methyl green	Nuclear	Green/turquoise

Fluorescence counterstains are often used with IHC too. DAPI (4', 6-diamidino-2-phenylindole) is a common nuclear dye, which generates a strong blue colour when bound to DNA and excitation is stimulated by ultraviolet light (UV). Alternatively, Phalloidin is a compound which binds filamentous actin (non-globular/free actin). It is normally conjugated with fluorophores to label the actin cytoskeletal structure, so the colour is dependent on the fluorophore which is used.

9. What is a good IHC control?

What should be a relatively simple process is choosing a negative control system to compare against. The Human Protein Atlas, along with the likes of Genecards.org and Uniprot.org are exceptional sources of pooled information, suitable for choosing appropriate IHC controls. Once you have a negative control you should be able to guarantee the specificity of your staining, but be aware that negative controls alone are not a suitable guarantee of antibody binding to the protein target of interest. In order to complete your analysis we recommend using knock-out or knock-down samples or a positive control tissue alongside to back-up your results. These should also be tested alongside a no-primary control and an isotype control.

10. Are my antibodies and reagents suitable?

Over the past few years antibody validation has been highlighted on numerous occasions in literature. A simple google search will pull up results from Nature reviews and methods detailing the importance both manufacturers and researchers have in assessing reagent quality. Characterise your antibody, and demonstrate both practicality and specificity for your experiment. Western blotting can often be carried out as a first line of specificity testing for a particular protein, as this can both quantify protein in your tissue, identify non-specific binding, and in combination with positive and negative controls can be validated for specificity of a particular protein. In many cases a polyclonal would be the most ideal to start a new immunohistochemistry protocol, because the variety of epitopes to which they bind allows for a stronger signal than that of a monoclonal antibody. But it’s not only the antibodies... You will need to check you buffer, the antigen retrieval solution and enzymes used. Carrying out a series of validation steps before initiating experimental stages will prove essential to prevent false positives or negatives occurring, and prevent waste on research funds.