In order to preserve the structural integrity and prevent tissue decay, samples must be fixed with a preserving agent and embedded in wax. The most common method of sample fixation uses a combination of paraformaldehyde (or neutral-buffered formalin) and paraffin wax, though special protocols may be required for certain other types of tissue. Once the tissue is prepared, thin slices of 5-20μm are made using a microtome, mounted to slides, and dried. Dried, paraffin-embedded slides are stable and can be stored at room temperature for extended periods (up to a year or more) before probing and imaging.

Once the researcher decides to probe and image a mounted section, they must first remove the paraffin wax to expose the tissue and allow antibodies to bind the antigen.

Alternatives to paraffin fixation

Frozen IHC

• For analysis of sensitive antigens that do not withstand the fixation process, such as nuclear proteins, enzymes, and DNA/RNA sequences (FISH)

• Requires flash freezing of tissues immediately after extraction and use of a cryostat for cutting sections

• Many enzymes retain function which can interfere with antibody detection without proper blocking steps

Free-floating IHC

• Allows analysis of larger tissue sections (~20μm), since antibody will penetrate from both sides of the sample

• While more handling is required by the researcher (transferring tissue sections between wash baths), the increase in section thickness provides more structural integrity

• Thicker sections provide more regional information, which can be useful for brain tissue in particular

The paraffin fixation process generates protein crosslinks, which mask certain epitopes from antibody binding. The antigen retrieval step is designed to break these linkages before the tissue is blocked and probed with antibodies.

There are two primary methods of antigen retrieval:

• Heat-mediated (Heat-induced epitope retrieval): uses a pressure cooker, microwave, or water bath; must be optimized for tissue type, as heat can dissociate some samples from microscope slide

• Enzyme-mediated: uses combination of citrate, Tris, HCl and EDTA buffers to chemically break linkages

Note: Heat-mediated antigen retrieval cannot be performed on frozen sections. However, since most frozen IHC protocols call for alcohol-based fixation steps, they eliminate the chance for crosslinking and therefore skip the antigen retrieval step entirely.

The blocking step has critical importance in IHC given chemical reactions that must take place before visualization of the antigen. Not only does this step prevent high affinity binding sites from yielding false positives by binding the primary antibody, but it also prevents native peroxidases and other enzymes from reacting with the substrates added in the final steps. It is important to consider what visualization technique(s) are planned before blocking, as extra steps will likely be needed for each substrate or reaction added to the protocol. While commercial blocking buffers are readily available, most times it should be sufficient to block using a dilution of normal serum (from the species of the secondary antibody) in PBS.

After blocking, the sample is incubated with primary antibody in solution. The dilution of the antibody is a crucial factor in determining the strength of the signal and in reducing background noise in the final image. It’s important to check the product data sheet for each antibody to ensure proper use.

Similarly to Western blotting, secondary antibodies for IHC-P or IHC-F are HRP (horseradish-peroxidase)-conjugated and are specifically selected for the species of origin of the primary antibody. After incubation with the secondary, slides are washed to remove excess antibody and then incubated with 3,3′-Diaminobenzidine (DAB) stain. DAB reacts chromatically with HRP to leave a brown stain wherever the secondary antibody is present. While the stain is less sensitive than some other methods (such as IF and WB) for detecting low-level antigen expression, it requires no special equipment (besides a standard light microscope) to view the results and is effective for assessing broad distribution of an antigen across tissue sections.

Since antibodies are designed to specifically detect the antigen of interest, most IHC protocols call for the addition of a counterstain to help visualize the overarching tissue structure. These stains detect common cellular features, such as nuclei, filaments, or organelles, allowing researchers to assess the relative expression of the target within and between different cell types. Depending on the type, the timing and method of applying a counterstain can vary, though many are applied as a final step immediately before sealing the slide.

Some of the most common counterstains:

• H&E (haematoxylin and eosin stain): haematoxylin is a dark violet/blue stain that marks the negatively charged DNA/RNA molecules of the nucleus, while eosin is a red/pink stain that marks primarily cytoplasmic components

• Nuclear Fast Red: a rapid, simple stain that marks nuclei with a pink color

• Methyl Green: a rapid, simple stain that marks nuclei with a blueish/green color

It is important to choose a counterstain opposite on the color spectrum from the color of the antigen signal. In addition to the chromatic stains listed here, there are a large number of fluorescent counterstains for IF experiments.