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  • IHC training
    Part 4: Troubleshooting


    Welcome to our training series on immunohistochemistry (IHC). We begin with essentials like sample preparation and antibody selection and then guide you through immunostaining protocols and troubleshooting.

    In this concluding part of IHC training, we share with you troubleshooting tips for some of the most commonly encountered problems before showing you some useful reagents, which could remove the need to troubleshoot.


    Part 4 overview

    4.1 No staining
    4.2 High background
    4.3 Incorrect staining
    4.4 Useful reagents to help with IHC troubleshooting
    4.5 IHC FAQs


    4.1 No staining

    One of the main issues you may come across in IHC is no staining. There are three main reasons why this may happen:

    1. Low/absent protein expression
    2. Poor tissue preparation
    3. Reagent/protocol issue

    Below you’ll find short videos, which go through tips to rectify these problems.

    Low/absent expression


    Poor sample preparation


    Reagent/protocol issue


    4.2 High background

    Another common problem with IHC staining is a high background. Our quick video below will highlight the major causes of high background and what can be done to solve this problem.


    4.3 Incorrect staining

    To find out about the third main problem with IHC, incorrect staining, please watch our short video below.

    For more troubleshooting tips, get the full guide for IHC troubleshooting.


    4.4 Useful reagents to help with IHC troubleshooting

    To help you with your IHC experiments, we have developed specific products that can reduce the amount of troubleshooting you’ll need to do.

    • Our recombinant RabMAb® antibodies offer increased sensitivity with no loss of specificity and the highest level of consistency between batches, making them ideal for demanding applications like IHC on paraffin-embedded tissues. They are also ideal reagents for customers using mouse models.

    Find out more about the advantages of our recombinant RabMAb® antibodies.

    • If you need to use a mouse monoclonal antibody on mouse tissues, there are several tips you can try to eliminate background staining.

    See our mouse-on-mouse (MOM) staining protocol.

    • You may also want to consider which secondary antibodies to use. Using an HRP-polymer secondary antibody will eliminate the endogenous biotin background in your IHC experiments. Also, HRP-polymer secondary antibodies are useful for the detection of low-expressing proteins as these antibodies use micropolymer technology to form smaller detection complexes that allow better tissue penetration and sensitivity. Besides, HRP-polymer secondaries bind more horseradish peroxidase than standard HRP secondary antibodies, increasing signal.

    Find the right secondary antibodies for your IHC staining.

    • If you are new to IHC, we recommend our optimized kits and reagents to ensure that you get the data you need in your first experiment.

    Find the right IHC kits and reagents.


    4.5 IHC FAQs

    Which antigen retrieval method is most suitable for this antibody?

    Any information 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 article 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 proteins; 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, as well as 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-treating 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 the 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 following the primary antibody incubation step.

    Which fixation method is most suitable for this antibody?
    The fixation and permeabilization methods used will depend on the epitope and the sensitivity of the antibody and may require some optimization. Fixation can be performed using cross-linking reagents, such as paraformaldehyde. These are better at preserving cell structures but may reduce the antigenicity of some cell components, as the crosslinking may 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 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 center 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?
    To examine the co-distribution of two (or more) different antigens in the same sample, a double or triple IHC 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.

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

    Alternatively, here are some key tips for multicolor IHC:

    1. The primary antibodies must be raised in different species to ensure each one can be detected by a separate secondary antibody.

    2. Each of the secondary antibodies used should be conjugated to a different fluorochrome or chromogenic chemical/enzyme. Each fluorochrome used should be excited by and emit a detectable wavelength with minimum overlap with one another. Chromogenic chemicals/enzymes used should each produce a different color. This will ensure that you can distinguish between the different target proteins in your sample.

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


    Summary

    And that concludes our IHC training series. We hope that you’re fully prepared to run your next IHC experiment and get those crisp and accurate IHC images. Now that you know the most common pitfalls with IHC procedure, you are likely to avoid them and, hopefully, won’t even need our troubleshooting guide.

    Once you feel ready, try taking our IHC quiz to test your new knowledge.

    Also, keep an eye on our main training page in case you need to brush up or get to get grips with other common applications in the lab.