Enzyme Substrates for IHC
There are a number of different substrates for the 2 main enzymes used in immunohistochemistry, but DAB (diaminobenzidine) as a substrate for horseradish peroxidase is by far the most commonly used. Although it is an excellent substrate, it is a carcinogen, and its brown colour might not be distinguishable from tissue elements such as melanin, and other tissue pigments.
There are a number of features of substrates that can be considered beyond the reporting enzyme & colour.
HRP Enzyme substrates AP Enzyme Substrates
It is important to choose the correct substrate for the enzyme present in your detection system. If you have the choice of which enzyme to use in your detection, consideration of any endogenous enzymes that may be present in your tissues and the properties desired in the enzyme substrate can determine which is best for your samples.
AP Detection Systems HRP Detection Systems
Format/ safety considerations
DAB is a known carcinogen, although it is undoubtedly one of the most versatile substrates available. Due to the chemical requirements to be a substrate, may of the colour variations have a similar chemical structure, but their status as carcinogens may not be fully understood. DAB powder or tablets may be a cheap option for a lab to prepare their own substrate however, using a ‘ready to dilute’ or ‘liquid format’ kits reduces the chances of inhalation of DAB or other substrate powders. These substrate kits are also often much more convenient and consistent.
The sensitivity of the method as a whole depends on a number of variables. More sensitive substrates can enhance the sensitivity of the detection system allowing further dilution of expensive reagents, such as primary antibodies. If endogenous enzymes can not be blocked adequately, nor avoided, then a less sensitive substrate might allow a better signal to noise ratio. Equally, if intending to do multiple labelling, differences in substrate sensitivity can be used to balance out the staining from a very prevalent antigen vs a weakly expressed target.
The best substrates for IHC tend to deposit most discreetly, giving crisply localised staining at the antigenic site. Some substrates do not deposit well (or at all) – and are more appropriate for ELISA – such as ABTS, TMB and pNPP. HRP substrates, in general, deposit slightly more crisply on sections compared to alkaline phosphatase substrates, so may be better for tissue staining such as neurones, where the structures are fine, or punctate staining.
Alkaline phosphatase substrates can be more translucent, so ideal where staining of prevalent antigens could otherwise obscure tissue morphology.
As described earlier, tissue pigmentation can be mistaken for DAB staining. There are a variety of colours available. Colour compatibility of substrates with the tissue and any counterstains to be used should be considered. Although choosing colour schemes can be nice, it can be secondary to the practicalities of the experiment, and how the substrate is to be visualised.
Colorimetric substrates suitable for IHC should all be visible under a light microscope.
Not all substrates are appropriate to use under darkfield microscopy conditions. If this visualization method is to be used to elucidate structures, the substrate needs to be suitable.
Some alkaline phosphatase substrates are also fluorescent if excited under the fluorescence microscope. This can be a useful feature, however the fluorescence excitation and emission is broad spectrum which is not normally ideal if using a number of different fluorochromes, unless bleed through can be compensated.
Tyramide fluorophores can be used in conjunction with HRP detection systems to yield a fluorescent endpoint.
For visualisation on an electron microscope, substrates must be electron dense. There are a number that have this property enabling multiple labelling and visualisation under the EM from a routinely stained section, rather than having to utilise a gold conjugate.
Taking into consideration any limitations of the visualisation system, and colour combinations including possible colour blindness in colleagues! Purple and red might not be as easy to differentiate between as the difference between brown and purple, although spectral imagers may cope with more minor variations in colour.
Dehydration & Mounting
Not all substrates are stable if incubated in alcohol, and these require aqueous mounting. Others will dissipate over time if left in an aqueous state, so require being dehydrated and mounted in a non aqueous media. It is most frustrating to have finished sections ready for viewing the following day, only to have found the staining has vanished overnight! The mounting requirements for all substrates and counterstains on any section should be the same – for instance Methyl Green counterstain requires non-aqueous mounting, however AEC substrate would dissolve in dehydration before the section could be visualised – so this is not a compatible combination.
Industrial methylated spirits are often used in place of ethanol as a cheaper alternative. This can affect the substrate after mounting, so when using new substrates or dehydration solutions do a test run to ensure these reagents are compatible.
Aqueous mountants Permanent/ organic mountants
Heat resistance is not normally a requirement for single labelling experiments however, primary antibodies used in multiple labelling experiments may require different conditions to bind effectively – one may be best used on an unmasked sample and the other on a section that has been unmasked at high temperature. Some detection kits used in multiple labelling can also benefit from a heating step to inactivate the first label before commencing the second label. In these instances, a substrate able to withstand the heating process is required.
Longevity of working solution
Substrate working solutions can have a short shelf life once prepared. Ideally working solutions should be prepared just before use, however substrates are being developed with automated stainers in mind, as a machine needs loading up with all solutions at the start of the protocol, and the substrate is always at the end!
The average run size you are expecting to do may influence the substrate you choose. As a rule of thumb, for most sections on a standard microscope slide, 100µl of substrate solution is sufficient for manual staining, but 200µl per slide for automated stainers. If you’re doing small runs of 10-15 slides daily, you may find that you throw away substrate solution every day. Although it may appear more expensive, a DAB designed for an automated stainer has a longer working solution shelf life and may enable you to make one working solution at the start of the week with less remainder. As DAB should be disposed of carefully, and in accordance with local regulations, the less to throw away the better!
Where colorimetric multiple labelling is being considered, it is important to select substrates compatible with each other, both chemically & by colour, as well as any counterstains being used. The localisation of the antigens to be stained is also a consideration – whilst there may be steric hindrance of the depositions of two substrates close together, and experienced researcher may be able to stain a section that the depositions of the 2 substrates offer a third colour demonstrating co-localisation. Immunoenzyme Multiple Staining Methods by Dr C.M. van der Loos (ISBN 1 85996 187 8) is a recommended read for colour mixing, however manufacturers of substrates may also provide guidance in their literature, such as this brochure on Multiple Labelling from Vector Laboratories.
In multiple labelling the order in which antigens and or substrates are applied can affect the outcome, so it is worth keeping an open mind as to the colour combinations of your antigens, it may be limited by practicalities.
It is possible to use 2 HRP or 2 AP enzymes in the same section with appropriate care, the advantage being the same detection system may be suitable for both labels, but all labels must be done sequentially and may benefit from a blocking step between each label protocol. (e.g. avidin/biotin blocking steps if using an ABC method, or potentially a heating step for polymer systems.)
Vector Laboratories have produced a handy guide to their enzyme substrates' properties.
*TMB can be used for immunohistochemistry, however it does not tend to deposit as discreetly as other HRP substrates and can be more variable. It requires a stabilizing solution to use for IHC as opposed to the non-precipitating version required for ELISA. Please contact our Technical Services department to discuss before you buy!