Immunohistochemistry (IHC)

IHC is an antibody-based technique used to detect antigens in fixed tissue sections and provides semi-quantitative data about target protein expression, distribution, and localization. Briefly, samples are fixed to preserve cellular integrity and incubated with blocking reagents to prevent non-specific binding of the antibodies. Samples are then stained with primary, then secondary antibodies, and the signal is visualized with a microscope.

The most challenging aspect of IHC is optimizing the experimental conditions required to generate a strong and specific signal for each antigen. Bio-Techne offers a variety of resources to streamline and troubleshoot your experiment to achieve the best results.

Sample Preparation and Fixation Resources

In IHC experiments, fixation is essential for preserving tissue morphology and maintaining the antigenicity of target molecules. Most common fixatives for preserving protein targets in tissues include aldehydes, alcohols and acetone. Tissue preservation can be achieved through paraffin embedding or cryopreservation (freezing). Often the preservation method is closely associated with the type of fixation. Formalin-fixed tissues are commonly paraffin-embedded following fixation, while frozen tissue sections are fixed with alcohol following cryopreservation.

Read more about these preparation methods and specific fixation techniques to effectively analyze tissue samples using IHC.

• IHC Sample Preparation (Frozen vs. Paraffin)
• Sample Fixation for IHC

Antigen (Epitope) Retrieval Protocols and Resources

While fixation is important for preserving tissue morphology, it can adversely affect the detection of epitopes in immunohistochemistry (IHC) experiments. For example, formalin fixation can lead to the masking of the epitope of interest and inhibit the binding of the primary antibody. Antigen retrieval encompasses a range of techniques employed to reverse epitope masking and restore antigen-antibody binding.

Learn effective antigen retrieval techniques:

• Heat-Induced Epitope Retrieval (HIER) Protocol
• Protease-induced Epitope Retrieval (PIER) Protocol
• Antigen Retrieval (Frozen Sections) Protocol
• Epitope Retrieval (HIER vs. PIER)

Blocking Non-Specific Binding

Overcome the challenge of non-specific interactions in IHC experiments by using a blocking reagent like normal serum or protein solutions, such as BSA.

• Know more about Blocking Non-Specific Binding in IHC

Antibody Selection and Detection Methods

Optimize primary antibody selection by considering protein characteristics, function, tissue, subcellular localization, and PTMs.

• Read tips on IHC Primary Antibody Selection & Optimization

Detection Methods: Antigen expression can be visualized in IHC using direct or indirect methods, generating chromogenic or fluorescent signals. Direct detection entails labeled primary antibodies, while indirect detection involves secondary antibodies. Both methods can be visualized through immunofluorescence (IF) or chromogenic reactions. For low-abundance antigens, signal amplification methods like biotin-based, HRP-polymer, or tyramide signal amplification (TSA) can be considered.

• Learn More about Detection Methods for IHC

Read Tips and Techniques for accurate results.

• IHC Troubleshooting Guide (FFPE)
• IHC Troubleshooting Guide (Frozen)

What is the difference between IHC and ICC?

IHC is the acronym for immunohistochemistry and ICC is for immunocytochemistry. IHC always refers to using 2-15 µm thick tissue sections cut either frozen or paraffin-embedded and mounted on histological slides. In addition, IHC may refer to staining 150 – 300 µm thick tissue sections incubated free-floating and only after that mounted onto histological slides for microscopy examination. ICC defines the technique of staining cells either in suspension or adhered to a matrix or cytosmears rather than tissue sections. IHC and ICC can utilize similar immunofluorescence and enzymatic staining protocols. 

What are the recommended working dilutions and volumes of primary antibodies?

There are different origins of primary antibodies developed for IHC applications including polyclonal, monoclonal, and recombinant antibodies. For most polyclonal antibodies, the working dilution range is from 1 to 15 µg/mL whereas for monoclonal and recombinant this range can be from 1 to 25 µg/mL. Polyclonal antibodies are used in lower concentrations to avoid non-specific background staining due to clones cross-reacting at higher concentrations with irrelevant tissue targets. Monoclonal and recombinant antibodies may be used at higher concentrations without the risk of producing background staining due to the lack of “contaminating” clones.

It is of critical importance to prepare antibody working solutions sufficient for staining tissue sections selected for the IHC experiment:

• Preparing more than needed will be a waste of primary antibodies and making less than needed may result in not covering entire tissue sections with antibody solution for incubation.
• Depending on the area of the tissue section it may be required from 15 to 200 µL of antibody solution per slide with attached tissue section.
• The tissue section should be completely covered with antibody solution to avoid the formation of dry spots resulting in inconsistent tissue staining.
• Adding an excessive amount of antibody solution to the tissue sections can cause leakage from the slide and result in weak or staining and even lack of staining in parts of the tissue section tissue staining.

To remove the guesswork from antibody solution calculations, it is usually recommended to use a calculating app (www.stainwell.com).

Which IHC controls are recommended?

IHC controls are always standard practice to make sure the staining signal is a result of the interaction of antibodies with its specific protein target rather than an artifact. Several types of controls can be used to address different issues. Before starting the IHC protocol it is always recommended to examine tissue sections under the microscope to assess the extent of tissue autofluorescence (if using immunofluorescence detection) and pigmentation (if using enzymatic chromogenic detection) which can obscure specific staining. For example, “aging pigment” lipofuscin, a product of oxidation of fatty acids, can be detected as yellow-brown speckles scattered throughout the cytoplasm resembling HRP converted DAB chromogen. Lipofuscin also has a very strong autofluorescence within a wide range (360–647 nm), emitting orange, green, yellow, and red light overlapping in color with many different fluorophores used for tissue detection. In addition, tissues may naturally autofluorescence which is caused by the presence of porphyrins and flavins.

1. No-primary antibody control: To rule out non-specific staining caused by the adsorption of secondary antibodies and detection reagents to tissue sections.
2. Primary antibody controls: These controls assess the specificity of primary antibodies.
   • Tissue controls: Positive control includes staining of tissues known to express the antigen of interest whereas negative control includes staining of tissues lacking the antigen. If there is staining in the positive and negative control tissue, it may be indicative of the primary antibodies cross-reacting with non-specific tissue targets.
   • Cell controls: Like tissue controls but utilize (a) negative and positive cells. (b) transfected cells, and (c) cell knockouts.
   • In Situ Hybridization (ISH) control: For the detection of mRNA coding the protein of interest. ISH can be combined with IHC for the co-detection of both mRNA and the protein to make sure both signals are present in the same cells.
   • Absorption controls: To confirm that antibody interacts with a specific amino acid sequence of the target protein. Primary antibodies are mixed with peptide immunogens and then this mixture is used for staining tissue sections. It is expected to see no staining because specific peptides bound to antibody binding sites block them from reacting with the target protein. The presence of a signal may be indicative that antibodies cross-react with irrelevant tissue targets.
   • Western Blot controls: To determine if antibodies are detecting the protein of correct molecular weight in lysates of the same type of tissues used for IHC staining. Seeing multiple bands of wrong size band may be indicative of the cross-reactivity of antibodies with irrelevant proteins.
   • Isotype controls: These controls are used to ensure staining is not a result of using the same antibody class. For example, if the primary antibody is IgG1, then using IgG of the same class 1 will serve as a control.