Counterstaining for Immunohistochemistry: Choices, Choices…

When it comes to immunohistochemistry (IHC) we spend a big chunk of time figuring out which antibody to use and which concentrations to try, but how many of us take the time to think about which counterstain would be the best choice for our tissue and the message we want to portray? Believe it or not, counterstaining can have a big impact on the final result. This little guide will introduce you to some of the stains/counterstains out there and hopefully open the door to a few more choices.

Where counterstains come in

As you probably know, most cells are colourless and transparent so no matter what your specific antibody is, whether it be fluorescent or not, it can be difficult to identify cell type and location. This is where counterstaining comes in. If you counterstain your sections post-immuno, it can help you to pinpoint the exact position of your positive cells. A counterstain also provides contrast so that the antibody-stained cells stand out more.

A vast array

There is a vast array of chromogenic and fluorescent dyes out there to fit every experimental design. These dyes can either be non-specific, staining most of the cells in the same way, or specific, selectively staining particular cellular compartments or chemical molecules within cells/tissues.

Colour me blue…purple….green: chromogenic counterstains

Haematoxylin, the product of the Haematoxylon Campechianum tree, is the most popular dye used in IHC and probably the go-to counterstain for most researchers. For more information on H and E staining, read Martins’ two articles here and here. It’s actually the oxidation product of hematoxylin (hematin) which constitutes the dye and not actually haematoxylin itself. There are a few different types of haematoxylin: Harris’s, Mayer’s, Carazzi’s, and Gill’s. These differ, in part, according to the oxidizing agent added e.g. mercuric oxide (Harris’s) or sodium iodate (Gill’s). Although for the most part mercuric reagents are now avoided.

Oxidized haematoxylin is usually combined with a mordant such as aluminium ions to form a metal-dye substrate which has a positive charge and can bind negatively charged chromatin. This can then stain the nuclei of mammalian cells a blue/purple by binding to lysine residues on nuclear histones, as opposed to other nuclear dyes that target the nucleic acids.

A popular choice

Despite its popularity, hematoxylin is not the only dye out there. Others that also target nucleic acids include methyl green and methylene blue. Methyl green is a blue/green nuclear stain and is also a great dye to use when you need to differentiate between DNA and RNA in tissues; when it is used as methyl green pyronin the methyl green stains the DNA and the pyronin stains the RNA.

Everything is blue

Methylene blue, an example of a cationic dye, is another widely used blue stain, but does not require a mordant. It gives a quick and simple nuclear counterstain and will bind to tissue anions such as carboxylic acid, sulphuric acid and phosphoric acid groups. These groups need to be ionized to bind the dye. Methylene blue will stain both the cytoplasm and the nucleus but to get maximal staining it is best to work in alkaline conditions. This can be helped with Löffler’s formula, which uses potassium hydroxide to raise the pH. This will ensure that the dye stains all of the proteins and the nucleic acids evenly so the whole tissue will appear blue and any differential staining will be eliminated.

From blue to pink

Although not so often used as a counterstain, another common blue dye is toluidine blue. This is a strong basic dye that stains nuclei a deep blue colour; however, it will also stain polysaccharadies a pink/red colour. This colour shift is called metachromasia, a term used when a dye stains a tissue component a different colour to the dye solution.

Binds to everything

Eosin can also be used as a counterstain when an antibody localized to the nucleus is used. Eosin is an anionic dye and works best in acidic conditions. It is mostly bound by ionized cationic groups of protein molecules, mainly the ?-amino group of the side-chain of lysine and the guanidino group of arginine. As nearly all proteins contain these two amino acids, eosin is bound by the majority of structures in any tissue.

Make me shine: counterstains for fluorescence

There is a whole range of fluorescent dyes/counterstains out there that can make for amazing and colourful images. As with chromogenic dyes and hematoxylin, there is a ‘golden standard’ for fluorescence: DAPI.

A golden standard

DAPI (4′, 6-diamidino-2-phenylindole) binds selectively to double-stranded DNA with no or very little cytoplasmic staining. Green or red ?uorescent labels can easily be used with DAPI, as it does not signi?cantly impair the signal. DAPI can be used on both ?xed and un?xed cytological preparations and tissue sections.


Hoechst dyes bind speci?cally to A/T-rich regions of double-stranded DNA, with no or very little cytoplasmic staining. Their characteristics are very similar to those of DAPI.  Both of these stains are great for most colours of primary/secondary antibody combinations. However, if a blue antibody needs to be used it would be better to use a red or green counterstain.

A popular red

Propidium iodide, a popular red dye, intercalates between DNA base pairs with little or no sequence preference and makes a good nuclear counterstain for use with green ?uorescent labels. Propidium iodide also binds to RNA, requiring treatment of specimens ?xed in aldehyde ?xatives with RNase in order to eliminate such staining.

Good choice for dead cells

SYTOX green is a high affinity nucleic acid stain that will penetrate cells with a compromised cell membrane, so it’s a good choice for sections or dead cells. SYBR green could be a good alternative to this. It preferentially binds double-stranded DNA over single stranded DNA and has good membrane permeability. As such it can be used in preparations of live cells.

Think about counterstains

This is, by no means, an exhaustive list of counterstains but rather an introduction to some of the choices out there. Hopefully this article will encourage you to think more about your counterstain and not just the steps that fall before so that you can generate some stunning images.

1 Comment

  1. Maricarmen Garcia on March 16, 2017 at 7:22 pm

    Great information!!!

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