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