Immunohistochemistry FAQs

Which antigen retrieval method is most suitable for this antibody?

Any information we have available on a suitable antigen retrieval method will be stated on the antibody datasheet.

If no information is available on the datasheet, we recommend starting with heat-mediated antigen retrieval using a citrate buffer. This will often require a certain amount of optimization by the end user. You may need to optimize the time of antigen retrieval, or try one of the other available methods. Visit our protocols page for more information on this and other antigen retrieval methods.

Which cell permeabilization method is most suitable for this antibody?

Solvents such as acetone and methanol are suitable for permeabilization when detecting intracellular protein; however, they are not compatible with all antibodies.

Detergents such as Triton or NP-40 will also partially dissolve the nuclear membrane and are therefore suitable when access to nuclear antigens is required. However, they are harsh detergents that can disrupt membrane proteins, especially if left on for too long and are therefore not suitable for detecting membrane proteins.

Tween 20, Saponin, Digitonin and Leucoperm are much milder membrane solubilizers. They will create pores large enough for antibodies to pass through without dissolving the plasma membrane. They are suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane, and are also suitable for soluble nuclear antigens.

​

Why should I block endogenous peroxidase activity? Do I use PBS or methanol to make the H₂O₂ solution?

​​​Endogenous peroxidases will react with the substrate solution (hydrogen peroxide and chromogen, eg DAB), leading to false positives. This non-specific background can be significantly reduced by pre-treatment of the sample with hydrogen peroxide before incubation with HRP-conjugated antibody.

Morphology of blood smears and peroxidase-rich tissues can sometimes be damaged by the hydrogen peroxide. Diluting the hydrogen peroxide in methanol is the best choice for fragile samples where preservation of morphology is required.

However, some cell surface protein markers are very sensitive to methanol or hydrogen peroxide quenching, reducing the staining of antigenic site, particularly on frozen sections. Using hydrogen peroxide in PBS is suggested for cell surface or membrane proteins. Another protocol modification is to quench with peroxide after first incubating section or cells with the primary antibody.

Which fixation method is most suitable for this antibody?

The fixation and permeabilization method used will depend on the epitope and the sensitivity of the antibody, and may require some optimization.

Fixation can be done using crosslinking reagents, such as paraformaldehyde. These are better at preserving cell structure, but may reduce the antigenicity of some cell components, as the crosslinking will obstruct antibody binding (antigen retrieval techniques may be required).

Another option is to use organic solvents such as methanol, ethanol and acetone. These remove lipids while dehydrating the cells. They also precipitate proteins on the cellular architecture.

The cells to be tested are growing in suspension. How can I stain these?

A suspension of bacterial cells or a cell line suspension can be spun onto a slide using a Cytospin™​ centrifuge. This will leave a small circle of cell sample adhered to the centre of the slide which can be dried, fixed and stained.

What is the excitation and emission wavelength of the fluorochrome?

A list of fluorochromes and their excitation and emission wavelengths can be found here.

Can I do double/triple staining? If so, what should I consider?

In order to be able to examine the co-distribution of two (or more) different antigens in the same sample, a double or triple immunofluorescence or immunohistochemistry procedure can be carried out. Primary antibodies against two or three different target proteins can be used either in parallel (in a mixture) or sequentially.

Save time with pre-optimised kits

We recommend our double staining and triple staining IHC detection kits for a stress-free experience. 

Alternatively, these are the key tips:

1. The primary antibodies must be raised in different species to ensure each one can be detected by separate secondary antibodies.
2. Each of the secondary antibodies used should be conjugated to a different fluorochrome or chromogenic chemical/enzyme. Each fluorochrome used should excite and emit a detectable wavelength with minimum overlap with each other. Chromogenic chemicals/enzymes used should each produce a different color. This will ensure that you can distinguish between the different target proteins.

We recommend optimizing the conditions for each antibody separately before trying them together.