Immunohistochemistry Procedure: A Guide to the 4 Main Steps (2023)

What Is Immunohistochemistry?

Immunohistochemistry is a favorite tool among clinicians to help diagnose a range of diseases by identifying abnormal cells, such as those in cancer.

In a nutshell, immunohistochemistry uses antibodies to detect proteins (antigens) that are specific to, or have altered expression in, abnormal cells within a tissue section (e.g. the liver, pancreas, or the heart). It can also be used as a predictor for treatment outcome (e.g. by demonstrating the expression of a target molecule for a particular drug).

In this article, we’ll outline the immunohistochemistry basics to get you off to a flying start with this technique.

Why Would You Want To Use Immunohistochemistry?

Immunohistochemistry isn’t just a useful clinical tool, it also has great applications as a basic research tool. It can provide you with a wealth of information on the expression of specific proteins within the context of tissue structure (rather than isolated individual cells).

This means that you can look at the location and colocalization of proteins within a cell, for instance in the nucleus, cytoplasm, or membrane, while the cell is still in its “natural” environment.

Got your attention? Great! It only gets better from here on in—not only is this a useful technique, but it is also pretty simple to perform!

Immunohistochemistry Basics: The 4 Main Steps

Ready for a rundown of the immunohistochemistry basics? A general immunohistochemistry protocol consists of four main steps:

  1. Fixation—to keep everything in its place.
  2. Antigen retrieval—to increase the availability of proteins for detection.
  3. Blocking—to minimize pesky background signals.
  4. Antibody labeling and visualization—to get the pretty pictures.

1. Tissue Fixation

This step is pretty important as itmaintains tissue structure and retains antigenicity (the availability of the antigens/proteins to be detected by antibodies). The fixation method you use depends on the tissue type you are using and your individual experimental requirements.

For instance, if you want good antigen expression but aren’t too fussed about the “look” (morphology) of your cells, opt for snap-frozen and acetone-fixed. However, if morphology is really important for you, go for the formalin-fixed and paraffin-embedded (FFPE) method.

(Video) Immunohistochemistry Protocol for Paraffin embedded Tissue Sections

2. Antigen Retrieval

If looksare what you are going for and you opt for the FFPE method, then you should consider pre-treatment with antigen retrieval agents. These improve antigen expression of your samples by breaking down formalin-induced antigen cross-linking, re-exposing epitopes on the antigen to antibody binding.

Heat and enzyme retrieval are both employed, with heat-induced epitope retrieval (HIER) now being the most commonly used. In simple terms, HIER involves heating your slides in buffer at pH6 or pH9 (depending on your antibody) using a microwave or a pressure cooker.

You can make your own buffers, but commercial reagents are also widely available for this purpose. Enzyme retrieval might be considered by some to be old hat, but still has its place.

You can use proteolytic enzymes such as pepsin or pronase to break down that cross-linking and expose the epitopes that your antibody needs to see; again, some antibodies are better suited to one method over the other.

3. Blocking

If you’ve ever performed a Western Blot you know that blocking endogenous materials before staining is crucial to minimize false-positive staining. For immunohistochemistry, there are two main factors to consider when blocking.

Block Endogenous Enzymes

First, you want to block or inactivate endogenous enzymes in the tissue that can activate substrates that might be used later in visualizing antibody binding (e.g. endogenous peroxidase and alkaline phosphatase).

Peroxidase is often an issue in tissues such as kidney or liver, and in those in which blood cells are present (arguably that means everything to some extent).

Alkaline phosphatase is particularly problematic in intestine and placenta (but much less so in FFPE material).

Be Aware of Endogenous Antibodies

Second, be aware of endogenous antibodies, for example, those on the surface of B lymphocytes in immune tissue; secondary antibodies may cross-react with these, leading to high background staining in your sample.

(Video) Immunohistochemistry Explained: Principle and Techniques for beginners

Choosing your secondary antibodies carefully may help here, but, in most cases, more general, non-specific binding arising from secondary antibodies needs to be reduced by a blocking step. Don’t fear, though, specific blocking approaches are available in each of these situations to improve results.

4. Antibody Labeling and Visualization

Now that you’ve done all the preparation, it’s time to get the pretty pictures. With immunohistochemistry, staining can be set up in one of two ways:as an indirect assay or using direct labeling.

Indirect Detection

With indirect detection, you use a secondary antibody with a covalently attached label. This secondary antibody binds to the primary antibody during the staining process.

Immunohistochemistry Procedure: A Guide to the 4 Main Steps (1)

Figure 1. Indirect staining methodology.

The standard indirect detection methodology can be seen inFigure 1but, in essence, there are two main steps to detect your antigen in this manner:

  1. Incubate your primary antibody (usually for 1 hour, but sometimes an overnight incubation may be necessary) on your tissue sample. This allows the antibody to bind to the antigen (assuming of course that the antigen is present). Once it is bound, you need to wash away any excess unbound primary antibody before incubating with a labeled secondary antibody.
  2. After another period of incubation (again 1 hour), excess secondary antibody is washed away and the amount of label associated with the primary antibody (i.e. indirectly via the secondary reagent) is quantified.

Direct Detection

This method is quicker and simpler than indirect detection, as the label is attached via a covalent bond directly to the primary antibody. This means you need only one incubation step and one round of washes (seeFigure 2). This method has several advantages over indirect detection, including decreased assay variability, reduced cost (by eliminating the need for secondary antibodies), and reduced time (as the second round of incubation and wash steps are removed).

Immunohistochemistry Procedure: A Guide to the 4 Main Steps (2)

Figure 2. Direct staining methodology.

Once you’ve labeled your samples, it’s time to visualize!

(Video) Immunohistochemistry | How to perform immunohistochemistry? | application of immunohistochemistry

If you’ve gone for fluorescent labels, you need to view your samples directly under a fluorescence microscope—remember to use the appropriate excitation wavelengths and filter sets based on the fluorophore you’ve used.

However, if you’ve opted for an enzyme label such as horseradish peroxidase, further incubation is required with the appropriate substrate. In these cases, the enzyme acts on the substrate to produce an insoluble colored component that is deposited at the site of antibody binding.

So Which Method is Best for Me?

Despite the obvious benefits of direct staining, the preparation of the directly labeled antibodies is often considered time-consuming and technically difficult. In addition, it is often thought that indirect staining is more sensitive than direct staining as multiple secondary antibodies are able to bind to one primary antibody, thus increasing the sensitivity of the target antigens.

However, during the binding of the secondary antibody and any wash steps, a proportion of the primary antibody will be dissociating from the antigen and, therefore, any amplification offered by the presence of the secondary antibody will be significantly reduced.

Essentially, you need to decide what is more beneficial for you. If time is of the essence and non-specific binding has been a problem in your immunohistochemistry experiments, then there are options available that allow you to directly label your primary antibodies quickly without any technical expertise required.

This handy table will help you decide which method is best for you:

MethodProsCons
DirectQuick methodology
Fewer reagents, cheaper
Non-specific binding eliminated

No cross-species reactivity

Dual staining is straightforward

Little signal amplification

Availability of directly conjugated antibodies for IHC staining is restricted

IndirectA small number of standard conjugated secondary antibodies is required

Commonly used technique

Non-specific binding may occur
Extra incubation and wash steps required

Dual staining difficult to achieve

We hope that this article has helped you with the immunohistochemistry basics! We’d love to hear how you get on with the techniques described; get in touch in the comments.

Struggling with unreliable results from your immunofluorescence? Download Bitesize Bio’s immunofluorescence troubleshooting guide poster—it explains the controls you need.

Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and tricks for all your histology experiments.

(Video) FFPE - Tissue Processing/Embedding/Sectioning for Histology, Immunohistochemistry (IHC), ISH & FISH

Originally published July 16, 2014. Reviewed and republished July 2021.

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Written by Innova Biosciences

Immunohistochemistry Procedure: A Guide to the 4 Main Steps (3)

(Video) Better IHC Step 1: Antigen Retrieval

FAQs

Immunohistochemistry Procedure: A Guide to the 4 Main Steps? ›

There are many critical steps in performing IHC. These include proper handling of the specimen, appropriate fixation, paraffin block preparation, antigen retrieval, selection and preparation of antibody and reagents, incubation, washing, and counterstaining [2].

What are the steps in immunohistochemistry? ›

There are many critical steps in performing IHC. These include proper handling of the specimen, appropriate fixation, paraffin block preparation, antigen retrieval, selection and preparation of antibody and reagents, incubation, washing, and counterstaining [2].

What are the four steps of immunocytochemistry? ›

The four steps of immunocytochemistry: (1) cell culture, (2) immunofluorescence staining, (3) confocal microscopy imaging, and (4) image analysis.

How do you perform an immunohistochemistry test? ›

To perform an IHC test, the care team must collect a tissue sample. The procedure to remove an abnormal or cancerous specimen is called a biopsy. Methods range from needle biopsies, which involve inserting a needle into the tumor, to excisional biopsies, which remove the entire tumor.

What is the preparation of immunohistochemistry? ›

Immunohistochemistry takes into account eight key steps that are involved in any immunohistochemistry preparation. It starts with sample collection, fixation, pre-treatment, blocking, incubation with your antibodies of interest washing, detection, and specimens preparation for imaging.

What is immunocytochemistry principle and procedure? ›

The steps involved in completing immunocytochemistry are seeding where the samples are applied to glass slides, fixation, and immunostaining where samples are secured and stained for visualization, using a microscope to view the samples, and then analyzing the images.

What is done in immunohistochemistry? ›

Immunohistochemistry (IHC) uses antibodies to detect antigens in a tissue sample. It's one lab technique a pathologist may use to check for signs of disease following a biopsy. IHC is commonly used to diagnose cancer, predict treatment response and determine likely outcomes (prognosis) of the disease.

What are the principles of immunohistochemical staining? ›

1)Primary antibody binds to specific antigen; 2)The antibody-antigen complex is formed by incubation with a secondary, enzyme-conjugated, antibody; 3)With presence of substrate and chromogen, the enzyme catalyzes to generate colored deposits at the sites of antibody-antigen binding.

How do you prepare immunohistochemistry slides? ›

Immunohistochemistry Protocol
  1. Immerse the slides in xylene (mixed isomers) 2 times for 10 minutes each.
  2. Immerse the slides in 100% ethanol 2 times for 10 minutes each.
  3. Immerse the slides in 95% ethanol for 5 minutes.
  4. Immerse the slides in 70% ethanol for 5 minutes.
  5. Immerse the slides in 50% ethanol for 5 minutes.

What are two detection methods used in immunohistochemistry? ›

In direct detection methods, the primary antibody is directly conjugated to a label. During indirect detection, the primary antibody is bound by a labeled secondary antibody that has been raised against the host species of the primary antibody.

How to do immunohistochemistry staining? ›

Immunohistochemistry Protocol
  1. Immerse the slides in xylene (mixed isomers) 2 times for 10 minutes each.
  2. Immerse the slides in 100% alcohol 2 times for 10 minutes each.
  3. Immerse the slides in 95% alcohol for 5 minutes.
  4. Immerse the slides in 70% alcohol for 5 minutes.
  5. Immerse the slides in 50% alcohol for 5 minutes.

What sample is used in immunohistochemistry? ›

Human and animal biopsies, or whole organs, are collected for preservation and IHC analysis, depending on the requirements of the researcher. Tissue must be rapidly preserved to prevent the breakdown of cellular protein and degradation of the normal tissue architecture.

What is the most commonly used antibody for immunohistochemistry? ›

The most commonly used is indirect detection with conjugated secondary antibodies. Here, a polyclonal antibody that reacts with immunoglobulins of the host species of the primary antibody is used for detection. The secondary antibody is covalently conjugated with molecular means of detection.

What are two detection methods used in ImmunoHistoChemistry? ›

In direct detection methods, the primary antibody is directly conjugated to a label. During indirect detection, the primary antibody is bound by a labeled secondary antibody that has been raised against the host species of the primary antibody.

How do you prepare ImmunoHistoChemistry slides? ›

Immunohistochemistry Protocol
  1. Immerse the slides in xylene (mixed isomers) 2 times for 10 minutes each.
  2. Immerse the slides in 100% ethanol 2 times for 10 minutes each.
  3. Immerse the slides in 95% ethanol for 5 minutes.
  4. Immerse the slides in 70% ethanol for 5 minutes.
  5. Immerse the slides in 50% ethanol for 5 minutes.

What is included in ImmunoHistoChemistry test? ›

IHC, or ImmunoHistoChemistry, is a special staining process performed on fresh or frozen breast cancer tissue removed during biopsy. IHC is used to show whether or not the cancer cells have HER2 receptors and/or hormone receptors on their surface. This information plays a critical role in treatment planning.

Videos

1. Immunohistochemistry IHC Tips and Techniques
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2. Better IHC Step 4: Chromogen
(Cell Signaling Technology, Inc.)
3. Mohs Surgery Immunohistochemistry: Step-by-Step Guide with Dane (4 of 4)
(Nathanial Miletta)
4. Immunohistochemistry Webinar: An Introduction to Immunohistochemistry
(Expedeon - now Abcam)
5. Mohs Surgery Immunohistochemistry: Step-by-Step Guide with Dane (1 of 4)
(Nathanial Miletta)
6. IHC for brain slice sections video protocol
(abcam)

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