Comfortable with the method, but searching for few extra tips? Guest Blogger Sarah Etheridge talks about her experiences of IHC experiments in the form of ten handy hints...
Immunohistochemistry, or IHC, allows you to visualize proteins in intact tissue retaining its microstructure, or as I like to say: ‘real life’. One of the advantages of this ‘real life’ visualization is that it allows for comparison between healthy and diseased tissues, thus the application is invaluable to both scientist and pathologist alike. The IHC protocol is straightforward, but contains many steps that may require initial optimization to ensure specific antibody binding and optimal visualization of the target protein. Here, I outline some tips for each of these steps and highlight which may need a little tweaking.
Tissue samples can be frozen or fixed. Freezing the sections generally maintains the conformation of the target antigen allowing superior antibody binding, but small ice crystals may form in the tissue [1, 2] and these sections may not be a good choice for long-term storage. Fixed and embedded tissue is a better alternative if you wish to hang on to your slides a little longer. The most common method of tissue fixation and embedding is formaldehyde fixation with paraffin embedding (FFPE). (It is possible to store FFPE embedded biopsies indefinitely at room temperature – making them an important resource for historical studies in medicine!).
Fixing tissue sections, before staining, with ice cold 50:50 methanol-acetone (MeAc) or 4% paraformaldehyde (PFA) may enhance antibody binding. MeAc, for example, dissolves cytoplasmic proteins allowing better visualization of membrane bound proteins.
Formaldehyde fixing causes cross-linking of proteins within the tissue, maintaining tissue morphology but denaturing the epitopes recognized by the antibody . Therefore, antigen retrieval is usually carried out before staining FFPE sections to unmask hidden or denatured target epitopes. This can be heat-induced or enzymatic [1, 4] with factors such as temperature, pH and time affecting antigen retrieval. It is a good idea to test a ‘battery’ or panel of antigen retrieval methods for each new antibody and tissue combination to determine optimal staining and rule out any non-specific background staining . A good starting point is boiling in citrate buffer at pH 6.0 in the microwave. Antigen retrieval is not needed for frozen sections, but the sections should be air dried for at least an hour prior to staining. De-waxing of FFPE sections in xylene and alcohol must be carried out prior to antigen retrieval and staining; following de-waxing, sections are rehydrated in decreasing levels of alcohol and then water, which leads me to my next top tip…
…Once sections are de-waxed and rehydrated it is important not to let them dry out! So use a specialized IHC staining tray to keep them in during incubations – many vendors sell these, they help keep tissue sections in a humidified environment. Alternatively, use a tray with tissue soaked in water. Top tip: Use a PAP pen– a special marking pen that provides a thin film-like hydrophobic barrier when drawn around a specimen – to keep solutions close to the sections once the permeabilization step has been carried out. Its use is not entirely essential but can be useful. (Don’t add PAP pen before permeabilization or MeAc fixation as it may be dissolved by Triton or MeAc and will bind to your tissue.)
For most proteins, it is a good idea to add a permeabilization step to your protocol. Permeabilization may not be necessary for transmembrane proteins whose epitopes are in the extracellular region. Permeabilization involves incubation with a detergent (e.g. 0.1% Triton-X100 in PBS). Detergents can also act as surfactants, breaking through some of the protein cross-linking that occurs during formaldehyde fixing, therefore aiding antibody binding to the correct epitope  and reducing non-specific hydrophobic interactions. Top tip: If staining isn’t working, try including detergent such as Triton at a lower level in all solutions (particularly for FFPE staining).
The blocking solution binds to non-specific binding sites within the tissue,  thus preventing non-specific binding of the primary and secondary antibodies to tissue components. Sections are incubated with an ‘innocuous protein solution’ such as bovine serum albumin (BSA) or serum from the host of the secondary antibody – I usually start with 3% BSA solution for 20-30 minutes. Proteintech also routinely uses 3% BSA, but can also use 5-10% goat serum as an alternative (as the secondary antibody it employs is raised in goat).
Your choice of primary antibody should depend on factors such as expression levels of the protein-of-interest and any other antibodies used in the case of co-localization. Antibodies from different species must be used in the case of the latter so that different secondary antibodies with unique conjugates can be used to distinguish between them. You must also choose whether a monoclonal or polyclonal is more suited to your needs; monoclonal antibodies give a highly specific signal whereas polyclonal antibodies can give a brighter one, particularly if expression levels of the protein of interest are low.
There is a chance of cross-reactivity and non-specific binding of other antibodies in the host serum. Purification techniques such as protein A/G purification, affinity purification or pre-absorption are used to prevent this from occurring. Proteintech affinity purifies all its antibodies with target antigen during product preparation; however, if you wish to carry out controls to test the specificity of primary antibody binding, a few options are set out below:
A guide for dilutions is usually provided with the antibody sheet, otherwise dilutions of 1 in 50 – 1 in 300 are good starting points for optimization; perhaps try a panel of primary antibody dilutions with a brand new antibody. Dilute the primary antibody in blocking solution (e.g. 3% BSA in PBS), then incubate for 1-2 hours at room temperature, or overnight at 39°F/4°C.
Wash off the primary antibody with wash solution such as PBS. A quick wash followed by three 5 minute washes should be sufficient. Frozen sections can be quite fragile, so drop solution slowly next to the section e.g. with a 1 ml pipette. Top tip: Alter the washing buffer to Tris-buffered Saline (TBS) rather than PBS.
Detection: Detection of the primary antibody is usually carried out with a secondary antibody directed against immunoglobulins of the host species of the primary antibody, conjugated to a fluorescent (e.g. FITC) or a chromogenic enzymatic tag (such as horse-radish peroxidase (HRP). Follow these tips for happy detecting!
For fluorescence experiments:
Don’t be intimidated by the many facets of an IHC protocol that could need optimization. Here’s a list of the first steps you should consider optimizing in the first stages of your IHC experiments:
1. Daneshtalab N, Doré JJ and Smeda JS, Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies., J Pharmacol Toxicol Methods., 2010;61(2):127-35.
2. Shi SR, Liu C, Pootrakul L et al., Evaluation of the value of frozen tissue section used as "gold standard" for immunohistochemistry., Am J Clin Pathol., 2008;129(3):358-66.
3. Fox CH, Johnson FB, Whiting J et al., Formaldehyde fixation. J Histochem Cytochem., 1985;33:845–853.
4. Yamashita S et al., Heat-induced antigen retrieval: mechanisms and application to histochemistry. Prog Histochem Cytochem., 2007;41:141-200.
5. Shi SR, Cote RJ and Taylor CR, Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem., 1997;45(3):327-43.
6. Saper CB and Sawchenko PE, Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry. J Comp Neurol., 2003;465:161–163.