How to Perform Immunohistochemistry

This How To Guide Will Cover

• IHC Theory Overview
• Working with Tissue Preparations
• Antigen Retrieval
• IHC Detection Methods
  • Direct Method
  • Indirect Method
  • PAP Method
  • ABC Method
  • Multiple Staining Method
• Counter Staining
• IHC Troubleshooting

IHC Theory Overview

Unlike ELISA and Western Blot, immunohistochemistry (IHC) has the ability to show where certain proteins are located within a tissue section at a fixed moment in time. But, just like ELISA and Western Blot, IHC uses antibodies to detect the presence of particular proteins.  Because this system uses antibodies, the inherent abilities and difficulties of working with antibodies still applies. IHC is the detection of proteins of interest, in a tissue section that has been "fixed" so that no chemical or biological changes occur after the point of extraction. Because the tissue is fixed, the proteins and other components of the cells and structure of the tissue are no longer moving, changing or interacting with one another. IHC does not denature the protein of interest, unlike standard Western Blots.  The protein is in its native, folded state, therefore certain antibodies that successfully recognize one protein in Western Blot, may not recognize the same protein in IHC. Antibodies reocognize only a single part of any protein (the epitope), and if that part is located inside the folded protein, then it is not accessible to the antibody and will not be detected. Be sure the antibody you use has been shown to detect the native form of the protein.

Working with Tissue Preparations

1. Samples can be prepared from the tissue of almost any organ in the animal body.

2. Properly fixing the organ is crucial. Fixing is normally done with paraformaldehyde, although some proteins are sensitive to aldehydes, in which case flash freezing should be performed. For paraformaldehyde fixation, transcardial perfusion should be performed immediately after death and before organ harvesting. This technique requires significant hands-on training, and will not be covered in this How to Guide. Once the organ has been harvested, it is usually embedded in paraffin. For flash freezing, the tissue is harvested as quickly as possible, and immersed in liquid nitrogen. Once the organ is frozen, it is usually embedded in a cryomold.

3. After the organ is fixed and embedded, it is sliced into thin layers, and those layers are then affixed to slides.  This step also takes a significant amount of training. If you do not have any experience in this step, you should send your samples to a histology department for slide mounting.

4. A deparaffinization step is required if your samples were placed in paraffin. It removes the paraffin still remaining on your slide, which would otherwise interfere with the ability of your antibodies to detect the proteins of interest.

Antigen Retrieval

• Proteins form cross-links during formalin fixation which make them difficult to detect. This step is not necessary for IHC, but strongly recommended for samples prepared with formalin, and should not be used on samples prepared using the flash freezing method.
• The method covered in this How to Guide is known as HIER (Heat Induced Epitope Retrieval). Prolonged heat exposure  breaks the protein bonds and improves the antibody's access to sites it recognizes (epitopes).

• The PIER method (Proteolytic Induced Epitope Retrieval) is not covered because it may result in destruction of the epitope site. However, it may be necessary if HIER is not successful.
• After almost every step, except serum blocking, a wash should be performed. This will remove unwanted proteins or chemicals.

Serum Blocking

Serum blocking is an important step. The presence of serum proteins keeps the antibodies from binding non-specifically to the slide.

Activity Blocking

Endogenous levels of certian enzymes, such as biotin and peroxidase will interfere with the reported signal from your antibodies. Performing a "block" to neutralize these enzymes is crucial, especially for the ABC and PAP methods, which will be covered shortly.

IHC Detection Methods

There are several methods of IHC detection, including the Direct Method, Indirect Method, PAP Method, ABC Method and Multiple Staining Method.  The following sections will cover all five of these ICH detection methods.  Washes should be performed after every step except after the serum blocking step, as you do not want to remove the blocking protein.

Direct Method

The Direct Method uses a conjugated primary antibody, and avoids further secondary antibody applications.  Visually there is nothing to distinguish staining using the direct method and other IHC detection methods, however signal strength will always be lowest using the direct method.

Indirect Method

The Indirect Method uses a primary antibody, and then a secondary antibody, which is conjugated and recognizes the primary antibody. The signal may be stronger, because multiple secondaries can recognize only one primary.  Signal strength is stronger with this method than the direct method, but it is otherwise indistinguisable.

PAP Method

The PAP (Peroxidase, anti-Peroxidase) Method uses a third antibody immunologically bound to peroxidase, which recognizes the unconjugated secondary antibody, improving specificity and signal. The use of this method allows the detection of proteins present in very small quantities.

ABC Method

The ABC (Avidin-Biotin Complex) Method uses an avidin peroxidase (or fluorescein) complex which recognizes a biotinylated secondary antibody. Visually, this staining method is indistinguisable from the indirect method.

Multiple Staining Method

This method uses multiple antibodies conjugated to different substrates or immunofluorescent dyes to detect more than one protein of interest.

In order to interpret your results, you must be familiar with the morphology of the tissue section that you are viewing. This takes considerable training and practice and will not be covered in this How to Guide. However, a common method is to locate landmarks through cell structure staining, also known as counter staining, which is covered in the next section.

Counter Staining

Many cell structures can be stained using dyes instead of antibodies. For example, the nucleus of cells can be identified using DAPI, which is a stain that binds strongly to DNA and fluoresces blue under UV light.  Other dyes are also available to stain cell structures, examples include:

• Crystal violet (Methyl violet) - stains cell walls purple.
• Commassie Blue - stains all protein blue.
• Ethidium Bromide - binds DNA and fluoresces orange.
• Haematoxylin - stains nuclein brown or dark purple.

IHC Troubleshooting

There are several commonly encountered issues when conducting immunohistochemical analysis.  Below are some of these common issues, as well as some troubleshooting techniques.  Have additional questions?  Email the Novus Technical Support Team at technical@novusbio.com.

• Problem #1: Poor or No Staining
  • Antibody dilution is too low
  • Antibody does not recognize native protein
  • Antibody is old or useless due to handling or freeze/thaw cycles
  • Deparaffinization was not thorough
  • Secondary antibody does not recognize primary antibody
  • Tissue was prepared incorrectly
  • Antigen retrieval was not performed
  • Proteins are sensitive to formalin fixation
• Problem #2: Non-Specific Staining
  • Primary or secondary antibody is staining non-specifically (there will be no recognizable pattern to the staining)
  • Washes were not thorough enough, or not often enough
  • Endogenous enzyme levels were not removed, therefore could have reacted with your detection substrate
  • Antibody used is made to the same animal that you are testing
• Problem #3: Signal is Too Strong
  • Antibody concentration was too high
  • Incubation time was too long
  • Detection substrate was applied for too long
  • Samples dried out