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Immunohistochemistry - Protocol & Troubleshooting

Immunohistochemistry (IHC) is a technique that detects antigens in tissue sections by immunological and chemical reactions. It uses specific binding between antibodies and antigens and chromogenic color development to detect and localize specific antigens in tissues. The technique is highly sensitive and specific and can detect a wide range of antigens in many animal species. Therefore, IHC is widely used in many research and clinical laboratories.

Creative Biolabs develops and optimizes IHC protocols, summarizes general IHC procedures for laboratory personnel, and provides troubleshooting and tips for each step of the process. In addition to basic guidelines for protocols, we also offer related products and services. In order for you to get more accurate and faster results, you can read the IHC protocol below before starting your experiment.

Solutions and Reagents

Stages Solutions and Reagents
Slide Preparation Tissue sections, wash buffer, fixative solution, antigen retrieval solution
Immunostaining Blocking buffer, primary antibody, labeled secondary antibody, dilution buffer, wash buffer, incubation buffer, chromogen
Counterstaining and Mounting Counterstains, wash buffer, mounting solution

Immunohistochemistry Procedure

Immunohistochemistry

1. Slide Preparation

Common tissue sections include paraffin-embedded (FFPE) sections and frozen sections. The first step is to slice on ice with a microtome, typically 4-7 μm thick, depending on the type of tissue. FFPE tissue sections need to be dried in preparation for dewaxing. Frozen sections can bedried or not, with optional steps depending on the type of section.

2. Antigen Retrieval

Antibody-binding epitopes may be masked in fixation. For this reason, antigen repair is sometimes required to reveal epitopes. Heat-induced epitope repair (HIER) is the most widely used antigen repair method, and a variety of heating methods are available to optimize antigenicity, such as microwaving and boiling.

3. Blocking

The blocking step can reduce unwanted background staining. Select the ideal blocking reagent and incubate the sample with the blocking buffer. Incubation time and temperature are set on a case-by-case basis. When finished, wash the slide and remove the blocking buffer.

4. Antibody Staining

IHC can be stained by indirect or direct labeling. For direct labeling, dilute the primary antibody appropriately and transfer the slide to the incubation buffer containing the primary antibody for incubation. Then wash the slides. For indirect assays, transfer the sections to an incubation buffer containing the secondary antibody and continue incubation. Wash the slide after completion to remove excess solution. Add the appropriate dilution of chromogenic agent to the slides and incubate away from light.

5. Counterstaining and Mounting

Counterstaining provides contrast with antibody staining to better distinguish the target signal. Select the ideal counterstains, incubate the sections, and rinse them upon completion. After all staining, use the appropriate sealing solution to mount coverslips and seal the samples. Mounted slides can be viewed under a microscope to observe the color of antibody staining in tissue sections.

Troubleshooting

There are multiple methods and combinations of IHC, so when poor staining results occur, there can be many reasons. We describe below possible scenarios and solutions that may be useful for troubleshooting. And hope you find them helpful in understanding immunohistochemistry procedures.

No or inadequate staining

  1. Antigen causes. If the test slide is inadequately stained or unstained, while the positive control slide is adequately stained, the antigen may be the problem. The antigen is not present in the test tissue or is present but at a level below the limit of detection. Consider using an amplification procedure or increasing the primary antibody concentration, incubation time, or temperature. If antigen coverage is caused by over-fixation or under-fixation of tissue sections, a modified antigen retrieval protocol is required.
  2. Antibody causes. If the positive control is weak or unstained and the test slide is also weak or unstained, the antibody may be the cause. Check the antibody, including conditions such as dilution concentration, incubation time, and temperature. Pay attention to check if the secondary antibody and primary antibody are compatible because the test requires the use of a secondary antibody that will interact with the primary antibody.
  3. Reagent causes. It is necessary to check the relevant reagents such as buffers and chromogenic agents, and expiration dates, storage parameters, pH, and order of use are all included.

High background

  1. Section causes. Consider first if the tissue section is too thick and thinner sections can be prepared. Then check if sections are adequately fixed or if necrosis and autolysis are present. If any of these conditions are present, avoid sampling necrotic areas and ensure that the tissue is properly fixed. As well, inappropriate kinds of or the excess use of section adhesives can affect the background. Throughout the process, check and ensure that tissue sections are not dry.
  2. Antigen retrieval causes. You may have used an inappropriate antigen retrieval method. Please re-evaluate the antigen retrieval conditions and optimize the antigenicity of your sample.
  3. Blocking causes. Possible reasons for this are inadequate blocking of endogenous enzyme activity, biotin, or protein during the blocking phase. You can increase the concentration of the blocking agent or use a different blocking agent. In addition, you need to be aware that the blocking buffer should be made from a blocking serum of the same species.
  4. Primary antibody causes. Improper antibody concentration causes high background. You can re-titrate the primary antibody and select the appropriate concentration. Check the primary antibody incubation time, too long time can also cause this problem. You can shorten the incubation time. Clean sections and no antibody solution remaining should be ensured as well.
  5. Secondary antibody causes. As with the primary antibody, check the secondary antibody and label concentration and incubation time. It is important to note that the secondary antibody chosen needs to be unbound to tissue immunoglobulins. Clean sections and no excess stain remaining should be ensured as well.
  6. Chromogen causes. If the chromogen concentration is too high and the reaction time is too long, you need to reduce the chromogen concentration and shorten the incubation time. It is also possible that the counterstain is masking the IHC reaction and we suggest you replace a different counterstain. As well as ensure that the sections are cleaned and no excess stain remains.

IHC is widely used in basic research. It can help you understand the distribution and localization of differentially expressed proteins in different parts of biological tissues. Not only is it a useful technique, but it is also very simple to perform. We hope that our products, services, and technical support will be useful to you in the conduct of immunohistochemistry.

You can contact us and learn more information, as well as tips and tricks, for immunohistochemistry experiments.

Reference

  1. Magaki S, et al. An Introduction to the Performance of Immunohistochemistry. Biobanking. Methods in Molecular Biology, 2019.
  2. Ramos-Vara J A. Principles and Methods of Immunohistochemistry. Drug Safety Evaluation. Methods in Molecular Biology, 2017.

For research use only. Not intended for any clinical use.

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