Tips and Techniques for Troubleshooting Immunohistochemistry (IHC)

• Specimen Preparation: The Foundation of Successful IHC
• Antigen Retrieval
• Staining
• Detection
• Analysis of Results: Fluorescent vs. Light Microscopy
• Troubleshooting Problems with IHC Results

Immunohistochemistry (IHC) is a technique in which antibodies are used to detect an antigen in sectioned tissue and is ubiquitous in the research, preclinical, and clinical settings for visualizing cellular components. This technique, which was first used in the 1940s, is widely used to increase understanding of the distribution and localization of differentially expressed proteins in tissues.

The steps for executing IHC and many of the tools and reagents have remained relatively unchanged for decades. However, understanding the basic steps in a simple IHC protocol is no guarantee of consistent, interpretable images. Optimization and fine-tuning are the keys to success in developing an IHC protocol for a new target.

The generalized steps of an IHC protocol are simple:

• Specimen Preparation
• Antigen Retrieval
• Blocking
• Primary Antibody Staining
• Detection

The steps seem simple, but optimization of your protocols and reagents can be the difference between no staining at all and bold staining that can alter the path of your research. Below, our R&D histologists and antibody validation scientists offer some insights into the optimizations that will help you avoid common IHC problems in order to get the most useful data and publishable images, regardless of your choice of technique.

Figure 1. Light microscopy image of anti-PIR antibody validation by IHC on FFPE human bladder sections using diaminobenzidine (DAB) enzymatic detection (brown chromogen) and counterstaining with hematoxylin (blue).

Figure 2. Fluorescent microscopy detection of non-phosphorylated rat neurofilament H (Cat. No. NE1023, green) by fluorescent staining of frozen sections of rat brain. Blue, Hoechst 33342.

Table 1. IHC sample preparation steps by tissue preservation method.

IHC Protocol Step	Frozen samples	FFPE samples
Tissue collection/fixation	Optional perfusion fixation	formalin
Sample embedding	freezing medium	paraffin
Tissue sectioning	cryostat	microtome
-20 °C storage	x
Deparaffinization/rehydration		x
Antigen retrieval		x
Endogenous enzyme blocking		x

Table 2. IHC staining (immunodetection) by analysis method.

IHC Protocol Step	Fluorescent IHC	Enzymatic detection for light microscopy
Wash slides in buffer prior to staining	x	x
Block to prevent nonspecific signal	x	x
Primary antibody incubation	Single primary, or primary cocktail	x
Wash sections 3x w/buffer changes	x	x
Incubate with secondary antibody	Fluorescent conjugate—can be a cocktail for multiple targets	x
Wash sections 3x w/buffer changes	x	x
Incubation with enzymatic substrate		x
Immerse slides in deionized water		x
Nuclear counterstain	DAPI or Hoechst (skip if included in mounting medium)	hematoxylin
Wash 2-3x in deionized water		x
Dehydration of sections		EtOH and xylene
Coverslipping with mounting medium	x	x
Store slides away from light	x

Specimen Preparation: The Foundation of Successful IHC

Regardless of preservation method or microscopic analysis approach, most histologists would agree that appropriate sample preparation, which preserves not only tissue morphology but also the target epitope(s), is the foundation for any successful IHC procedure. The protocol varies significantly depending on whether analysis by fluorescence or light microscopy is planned.

Tissue Collection and Fixation

Appropriate and efficient tissue collection and preservation for IHC safeguards cell and tissue morphology while halting proteolytic and microbial degradation. The two principal methods for tissue preservation for IHC are rapid freezing and FFPE (formalin fixed, paraffin embedded).

In frozen tissue preparations, tissue is rapidly harvested into a nonaqueous medium, and subsequently sectioned at very cold temperatures on a cryostat. FFPE preparations offer the convenience of indefinite storage and sectioning of tissue embedded in paraffin blocks at room temperature on a microtome.

Cryopreservation

Immediately following dissection, fresh tissue should be quickly frozen in chilled isopentane (2-methylbutane) or an equivalent alternative. Rapid freezing is essential to prevent the formation of ice crystals that can damage tissue. Frozen tissue is then stored at -80 °C until sectioned. For large tissues such as human organs, tissue must first be sectioned into smaller blocks to facilitate faster freezing that is uniform throughout the tissue volume.

After thawing and prior to cutting, frozen tissue is embedded in molds with a freezing medium which is also used to adhere it to the cryostat chuck and allow the frozen tissue to contact the sectioning blade in a controlled manner. Tissue should also be allowed to equilibrate in the cryostat chamber for 30 minutes prior to cutting to facilitate the acquisition of consistent and representative tissue slices. The optimal cryostat temperature for sectioning depends on the tissue type and freezing protocol. Be mindful to keep track of tissue orientation during all stages of cryostat sectioning. After each section is made, gently lay the section out on a frozen glass slide. The use of a frozen paintbrush and an anti-roll tool (along with lots of practice) will help avoid tissue section rolling, folding, wrinkling, and tearing.

Antigen Retrieval

Although FFPE is an effective and reliable method for ensuring the preservation of tissue morphology for extended periods at ambient temperatures, this format simultaneously renders many target epitopes inaccessible to antibody detection. Antigens in FFPE tissue sections may be unmasked by one of these retrieval methods:

• HIER (heat-induced epitope retrieval): Tissue sections on slides are subjected to a specific sequence of heating with simultaneous pressure increase in a citrate buffer. This is achieved in a device similar to a pressure cooker
• PIER (protease-induced epitope retrieval---also called EIER, enzyme-induced epitope retrieval) refers to the employment of an enzyme—proteinase K, trypsin, or pepsin—incubated at 37 °C.  TIP: For a few slides, the enzyme solution can be added directly to slides; in most cases, however, reagent concentration, temperature and incubation time are best controlled by simultaneous immersion of all slides to be processed in a bath containing the enzyme.

Staining

Selecting Antibodies for IHC

Specificity: Primary antibody selection is a critical step for effective IHC. One especially important consideration is antibody specificity, which ensures that the antibody binds only to the protein of interest. A gold standard for assessing specificity is the finding that staining is absent in tissue from which the protein of interest has been knocked out, although other methods, such as the presence of a single band using Western blotting, also exist. Selection of a secondary antibody should always ensure that the secondary is specific to the species that the primary antibody was originally obtained from. Several additional factors can each determine the efficacy of the secondary antibody and include, epitope specificity to the primary antibody, purification methods, and if the antibody is absorbed against cross-reactive immunoglobulins.

Application suitability: Reputable antibody suppliers will typically indicate immunodetection applications for which an antibody has been validated, and should supply data to demonstrate results for IHC/ICC (immunocytochemistry). For example, antibodies that successfully label an epitope under the denatured conditions of Western blotting may not necessarily perform during IHC, in which the native protein conformation is often preserved.

Clonality: Monoclonal antibodies are derived from expanding a single plasma cell, and therefore recognize a unique epitope. While monoclonal antibodies offer specificity, polyclonal antibodies can be a better choice when the protein target is expressed at low levels, as they recognize multiple epitopes and therefore offer greater sensitivity.  Conversely, the manufacturing process for polyclonal antibodies makes them intrinsically subject to greater lot-to-lot variation within product specifications.

Host source: A fourth important antibody consideration is the host species that was used to generate the antibody. To avoid cross-reactivity, it is essential that the antibody be raised in a different tissue than that of the tissue under study.  The primary antibody host species will also need to be matched to the secondary antibody during detection.

Blocking: Non-specific binding of the antibody is of paramount concern when it comes to immunodetection of the antigen by the primary antibody.  However, non-specific detection of antigens by the secondary can also lead to significant background after the substrate addition step.  A critical step to reduce background staining and ensure antigen specificity is by incorporating a blocking step prior to the addition of the primary antibody. The blocking solution should ideally contain serum that matches the species of the secondary antibody.

Detection

Detection is typically achieved using one of two methods: (a) colorimetric or enzyme-mediated detection and (b) fluorescence-based detection.

In the colorimetric method, the bound primary or secondary antibody is conjugated to a substrate which yields a precipitating product when converted by an enzyme. This precipitate is visible as colored staining when viewed by light microscopy.

In the fluorescence-based detection method, antibody bound to the antigen of interest in the tissue is directly or indirectly conjugated to a fluorophore (also sometimes called a fluorochrome), a molecule that fluoresces in the presence of light of a specific wavelength.

Analysis of Results: Fluorescent vs. Light Microscopy

Fluorescent Microscopy

Both immunofluorescent ICC and IHC require an understanding of the configuration of epifluorescence or confocal microscope that will be used to analyze the sample. The best results will be obtained when spectral characteristics of the fluorophores conjugated to the primary or secondary antibody are matched to the excitation source (usually a laser) and emission filters of the available microscope. For more about designing a fluorescence microscopy analysis experiment, visit this page.

Light Microscopy

White light or brightfield microscopy may be more accessible to researchers as the necessary equipment is available in most labs, but is limited by the number of targets which may be simultaneously detected in the same tissue section.  This is because deposition of the chromogen at the site of antibody binding to antigen in the tissue is dependent on enzyme-substrate activity, and the most common, reliable reagents (such as diaminobenzidine, or DAB, and alkaline phosphatase, or AP) do not offer multispectral colorimetric detection that would permit simultaneous detection of multiple targets.

Troubleshooting Problems with IHC Results

The following table presents some common problems encountered with IHC results, along with possible explanations and suggestions for technical correction/optimization.

Problem	Possible Cause	Solution
Poor or no staining	Antigen not present in tissue sample	Review the literature to confirm that the target is expressed in your sample tissue. Or, check mRNA expression by in situ hybridization.
Poor or no staining	Antibody degradation	Aliquot and store antibodies in smaller volumes to minimize repeated freeze-thaw cycles and exposure to warming and light (for fluorescent antibodies). Store as recommended by the manufacturer.
Poor or no staining	Insufficient primary antibody bound to sample	Use more concentrated primary; increase incubation time
Poor or no staining	Inadequate tissue fixation	Ensure that tissue is fixed immediately after collection with fixative at the appropriate concentration and temperature, and for sufficient duration.
Poor or no staining	Inaccessibility of antibody to antigen due to overfixation or fixing with inappropriate fixative for tissue or target	Reduce duration of fixation; try fixing tissue at 4 °C and increase duration, instead of shorter fixation at ambient T.
Poor or no staining	Primary & secondary antibodies not compatible	Verify that the secondary antibody that is specific for the primary antibody host species
Poor or no staining	Defective detection reagents	Prepare fresh enzymatic or other reagents.
Poor or no staining	Enzyme-substrate reactivity; improper pH of substrate buffer	Deionized water may contain peroxidase inhibitors that can reduce enzyme activity. Use buffer in the recommended pH range for specific substrates.
Poor or no staining	Primary antibody may not be suitable for immunohistochemistry application	Check data sheet or vendor website for recommended applications and supporting data
Poor or no staining	Deparaffinization was not sufficient	Deparaffinize sections longer using fresh xylene.
Poor or no staining	Ineffective antigen retrieval	Verify heat and pressure conditions for HIER, reagent integrity for PIER; or, try a different retrieval method.
Poor or no staining	Reagents omitted or not applied in order	Repeat procedure with careful attention to protocol
Poor or no staining	Antibody diluted in incompatible buffer---for example, some monoclonals may be unable to bind target due to ionic strength of dilution buffer	TBST may be more universally compatible than PBS, except for in fluorescent applications Evaluate staining on frozen tissue with different fixatives (EtOH, acetone, etc)
Poor or no staining	FFPE tissue incompatible with antibody	Avoid azide, which is HRP incompatible, in buffers. TBST is not compatible with AP detection.
Poor or no staining	Buffer incompatible with enzyme	pH of retrieval buffer in HIER must be optimized for the target. Low pH can be better for nuclear targets, high pH for cytoplasmic / membranous targets.
Poor or no staining	Antigen retrieval is inappropriate	Some targets, particularly glycoprotein or collagen targets, may benefit from PIER and / or HIER together. Shorten or eliminate retrieval, as HIER or PIER may destroy or alter the target itself in rare cases. When HIER or PIER cannot be used, try permeabilization instead (ie. 0.3-0.5% Triton X-100 ), before blocking.
Poor or no staining	Peroxidase block is inhibiting staining of target	Move peroxidase block step to after primary incubation (may be necessary for certain CD markers).
Overstained Tissue	Primary and/or secondary antibody concentration is too high	Determine optimal primary antibody concentration by staining adjacent sections, varying only the primary antibody concentration.
Overstained Tissue	Secondary incubation period is too long	Secondary antibody incubations >60 minutes will typically not enhance signal, and may lead to undesired Optimize the duration with each component: antibody, substrate, enzyme etc.
Overstained Tissue	Nonspecific binding of primary and/or secondary reagents to tissues	Treat tissues to minimize or block nonspecific binding.
High Background/Non-specific binding	Tissues may have high levels of endogenous molecules that are also present in incubation mixtures. For example, peroxidase in blood cells may remain in tissues.	Try incubating with normal serum from species other than the source of primary antibody. For tissues containing interfering peroxidase: treat tissue with 3% H2O2 or 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature.
High Background/Non-specific binding	Tissue not washed properly	Wash at least 3x with phosphate buffered saline (PBS) for 2-5 minutes after each staining reagent step
High Background/Non-specific binding	Secondary antibody cross-reactivity or nonspecific binding	The secondary antibody may show a strong or moderate affinity for the tissue itself. For example, egg whites, sometimes used to coat slides, contain high amounts of avidin. Avoid using egg whites to prevent avidin from binding biotinylated secondary antibody during staining.
High Background/Non-specific binding	Tissues sections may have dried out at some point in the protocol	Keep the sections wet at all times during the IHC protocol
High Background/Non-specific binding	High level of endogenous peroxidases or phosphatases	Use an appropriate inhibitor to block peroxidase or phosphatase activity, which can interfere with HRP and AP enzymatic chromogen methods, respectively
High Background/Non-specific binding	The primary antibody host species is the same species as the tissue samples	Select a primary antibody raised in a host other than the species of the tissues you wish to analyze by IHC
Altered Tissue Morphology	Antigen retrieval technique was too harsh	Try a different method or optimize method to make heat or enzymatic conditions less harsh
Altered Tissue Morphology	Frozen sections detach from slide	Increase fixation time. Use freshly prepared slides
Altered Tissue Morphology	No resolution of sections at the single-cell layer level.	Prepare thinner sections
Altered Tissue Morphology	Tissue is not intact/appears damaged	Increase fixation time; use smaller pieces of tissue. Fixative must have access to the tissue interior.