Immunohistochemistry with Vector Laboratories

Tissues

Tissues used are most commonly FFPE or frozen sections. Not all primary antibodies will work on both tissue types, and some antigens can be destroyed by fixation methods, so where possible start by matching your fixation to your primary antibody’s needs.  

Further information on Sample preparation can be found in this guide.

Primary antibodies 

As mentioned in "Tissues", ensure that your primary antibody is known to recognise your antigen in your type of fixed tissue.

You should also ideally choose a primary antibody of a different species to your tissue if possible. If you can’t, you may need to consider a conjugated primary antibody, or a kit designed to eliminate cross reactivity. Our Technical Team are happy to discuss your case.

Antigen Unmasking  

Some antigens need unmasking to enable the primary antibody to recognise them. Techniques such as enzyme digestion or Antigen Unmasking/ Heat Induced Epitope Retrieval (HIER) are often used. This step is usually dictated by the primary antibody to be used and is most commonly required on FFPE samples.

Secondary antibodies  

Secondary antibodies offer a flexible way to detect a primary antibody, and also help to increase the sensitivity of the method, although they can also contribute to background staining, especially if the primary antibody is closely related to the tissue species – e.g. mouse and rat. The secondary antibody should be chosen to detect the primary antibody, and antibodies adsorbed against the tissue species are recommended if there’s a cross reactivity issue. The secondary antibody needs to either have a hapten, such as biotin or the enzyme on.

Mounting  

The best mounting media will be determined by your substrate and counterstain. Some substrates can be mounted in an aqueous media, however others require dehydration and mounting in an organic based solvent, or ‘permanent’ mounting.  

Detection

Kits such as the ImmPRESS® kit are essentially secondary antibodies conjugated to an enzyme-laden micropolymer backbone. This offers the increased sensitivity of a VECTASTAIN® ABC kit, but in a shorter method and is also a biotin free method.  

The ABC kits are exceptionally flexible and can be used after any biotinylated reagent, which can be useful for primary antibodies made by more unusual species, such as chicken or guinea pig etc, as well as the traditional mouse, rabbit or rat. It is a longer method and endogenous biotin may need to be blocked to prevent background, particularly in tissues such as liver and kidney that are naturally high in biotin.

These kits can come either using HRP or AP as the reporting enzyme, these can also be endogenous in tissues, and may require blocking, or where the levels are very high, an alternative reporting enzyme can be considered.

Substrates

It is important to buy a substrate to match the enzyme of your detection kit. For HRP detection, substrates such as DAB, Vector® NovaRed and Vector® VIP are popular. For AP Vector®Red is a good chromogenic and fluorescent substrate. ImmPACT substrates are more sensitive and may have other benefits compared to their non-ImmPACT counterparts.  

The Enzyme Substrates brochure has further details on the properties of Vector Laboratories substrates.  

If you intend to add a counterstain to your tissue, it is important to consider compatibility, both with the substrate colour and the way the substrate and counterstain require mounting.