The counterstain can help to add structural definition to immunohistochemical stains to enable more accurate information on localization, morphology and cell structure within the tissue section. Nuclear counterstains are the most popular, as cytoplasmic staining can be heavy and which may mask a lighter antigen label. Dependent on using a progressive or regressive counterstain, the initial incubation time can be an important variable to control to achieve a light counterstain.
Where multiple antigens have been labelled, it may be that a counterstain is not necessary, and may even be distracting. As examples, many of the multiple labelling images from Vector Laboratories do not use a counterstain.
Counterstains have to be compatible with the enzyme substrate used; not only to ensure a good contrast of colour, but will also need to be compatible with the mounting requirements.
These single stains are used in a number of other histological special stains. Typically the concentration of these solutions for immunohistochemistry staining may be higher than when they are used in special stains.
There are several formulations of Hematoxylin, with Gill’s and Mayer’s being most popular for immunohistochemistry.
A progressive stain that requires a blueing step - usually ammonium hydroxide in either alcohol or water, depending on the enzyme substrate’s solubility in alcohols.
A regressive staining formula; sections are incubated briefly with Mayer’s hematoxylin then rinsed, removing any excess staining.
Both varieties of hematoxylin provide good contrast with most enzyme substrates, and can be mounted either aqueously or non-aqueously.
A useful alternative green counterstain where the blue of hematoxylin is not compatible with combinations of enzyme substrates selected. Methyl green needs heat to be able to stain, either warming to around 60ºC in a waterbath for batch processing, or on a slide warmer for individual slide processing. Methyl green counterstain also needs to be differentiated in acetic acid, that can affect the colours of some enzyme substrates.
Requires non-aqueous mounting, and is therefore not compatible with enzyme substrates such as AEC that are soluble in alcohols.
Nuclear Fast Red (also known as Kernechtrot) has a more limited range of enzyme substrates that it demonstrates excellent colour contrast with, but is a useful alternative to hematoxylin that can also be aqueously or non-aqueously mounted.