In immunohistochemical techniques, there are several steps prior to the final staining of the tissue antigen, and many potential problems can affect the outcome of the procedure. This article discusses the major problem areas in IHC staining.

Example of IHC staining

The following image provides an example of IHC staining.

Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) cancer tissue. Analysis was performed to compare Connexin 43 membrane staining in FFPE sections of human lung adenocarcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, heat-induced epitope retrieval (HIER) was performed using 10 mM sodium citrate (pH 6.0), followed by heating in a microwave for 8 to 15 minutes. After HIER, tissues were blocked in 3% H2O2 in methanol for 15 minutes at room temperature, washed with distilled H2O and PBS, and then probed overnight at 4°C in a humid environment with an Invitrogen Connexin 43 monoclonal antibody (Cat. # 13-8300), diluted 1:20 in PBS/3% (w/v) BSA. Tissues were washed extensively in PBS buffer containing 0.05% (v/v) Tween-20 (PBST). Detection was performed using an HRP-conjugated secondary antibody followed by chromogenic detection using DAB as the substrate. The sections were counterstained with hematoxylin and dehydrated with ethanol and xylene prior to mounting.


Strong background staining

The following points are provided to help identify the cause of high background staining, which results in a poor signal-to-noise ratio. See also the additional notes sections at the bottom of this page for more information.

Cause: Endogenous enzymes

Incubate a test tissue sample with the detection substrate alone for a length of time equal to that of the antibody incubation. A strong background signal suggests interference from endogenous peroxidases or phosphatases.

Endogenous phosphatases can be inhibited with the endogenous alkaline phosphatase inhibitor, levamisole. 

Cause: Endogenous biotin or lectins 

High background can occur when endogenous biotin is not blocked prior to adding the avidin–biotin–enzyme complex.

  • Solution: Follow the steps in Tech Tip #16: Block endogenous biotin.

If the ABC complex is made with avidin, the highly-glycosylated protein can bind to lectins in the tissue sample.

  • Solution: Block endogenous lectins with 0.2 M alpha-methyl mannoside in dilution buffer. Alternatively, use streptavidin or Thermo Scientific NeutrAvidin Protein instead of avidin, because both are not glycosylated and won't bind to lectins.

Cause: Secondary antibody cross-reactivity or nonspecific binding

The secondary antibody may show a strong or moderate affinity for identical or similar epitopes on non-target antigens.

  • Solution: If normal serum from the source species for the secondary antibody is used to block the tissue, then increase the serum concentration to as high as 10% (v/v), if necessary. If you are blocking with another reagent (BSA, nonfat dry milk), then add 2% (v/v) or more normal serum from the source species for the secondary antibody. Alternatively, reduce the concentration of the biotinylated secondary antibody.

Egg white, which contains avidin, was often used to coat slides, dilute antibodies or block tissue samples because it is a readily available and inexpensive source of carrier proteins. It is used very rarely nowadays.

  • Solution: Avoid using egg whites to prevent egg white–based avidin from binding biotinylated secondary antibody during IHC staining. Synthetic tissue adhesives as well as avidin-free antibody diluents and blocking buffers are readily available.

Cause: Issues with the primary antibody 

Nonspecific interactions between the primary antibody and non-target epitopes in the tissue sample occur regularly during incubation but at a level that does not influence background staining. A high primary antibody concentration will increase these interactions and thus increase nonspecific binding and background staining.

  • Solution: Reduce the final concentration of the primary antibody used for staining.

The primary antibody may also show a strong or moderate affinity for identical or similar epitopes on non-target antigens.

  • Solution: Increase the blocking buffer composition and/or concentration, or use a different primary antibody.

The primary antibody diluent may contain little or no NaCl, which helps to reduce ionic interactions.

  • Solution: Add NaCl to the blocking buffer/antibody diluent so that the final concentration is between 0.15 M and 0.6 M NaCl. The best NaCl concentration to use will have to be determined empirically.

Weak target staining

See also the additional notes sections at the bottom of this page for more information.

Cause: Enzyme–substrate reactivity 

Even when the tissue sample is properly prepared and labeled, the enzyme–substrate reaction must occur for the chromogenic precipitate to form. Deionized water can sometimes contain peroxidase inhibitors that can significantly impair enzyme activity. Additionally, buffers containing sodium azide should not be used in the presence of HRP. Finally the pH of the substrate buffer must be appropriate for that specific substrate.

A simple test to verify that the enzyme and substrate are reacting properly is to place a drop of the enzyme onto a piece of nitrocellulose and then immediately dip it into the prepared substrate. If the enzyme and substrate are reacting properly, a colored spot should form on the nitrocellulose.

  • Solution: Change the enzyme diluent and/or prepare substrate at the proper pH and repeat the test.

Cause: Primary antibody potency 

Primary antibodies generally lose affinity for the target antigen over time, either due to protein degradation or denaturation caused by long-term storage, microbial contamination, changes in pH or harsh treatments (e.g., freeze/thaw cycles).

Test the primary antibody for potency by staining tissue samples known to contain the target antigen with various concentrations of the primary antibody; do the test concurrently with the test sample. If the positive control is not positive for the target antigen at all, then this suggests that the primary antibody has lost potency. In fact, it is good laboratory practice to always run a positive control sample through your staining protocol along with the experimental samples.

  • Solution: Ensure that the antibody diluent pH is within the specified range for optimum antibody binding (7.0 to 8.2) and that the antibody is stored according to the manufacturer's instructions. To prevent contamination of your antibody solutions, wear gloves when dispensing antibodies, and use sterile pipette tips, if appropriate. Even if you store your antibodies in a refrigerator, always divide them into separate small aliquots. This prevents contamination or loss of the whole vial of antibody if a problem arises.

Cause: Secondary antibody inhibition

While high concentrations of the secondary antibody can increase background staining, extremely high concentrations can have the opposite effect and reduce antigen detection.

To test if the secondary antibody concentration is inhibitory, stain positive control samples using decreasing concentrations of the secondary antibody. An increase in signal as the concentration decreases suggests that antibody concentration is too high.

  • Solution: Reduce the concentration of the secondary antibody.

If the diluent and/or blocking solution contains antigen-neutralizing antibodies, such as those found in serum, then the antibodies will block secondary antibody binding.

  • Solution: Remove the neutralizing antibodies or change to a different diluent and/or blocking solution.


Autofluorescence

If a fluorescent marker is being used, check to make sure that there is no autofluorescence in the unprocessed, fixed tissue. In particular, FFPE sections often show strong autofluorescence that may be difficult to inhibit. Many of the options listed above can then be tested to identify the cause of autofluorescence.

If there is autofluorescence in the test sample, then this suggests that either the tissue sample shows inherent autofluorescence (which is common) or that the fixation method is causing the sample to autofluoresce. To determine if the fixation step is the cause of the autofluorescence, test different fixatives (i.e., if aldehyde fixation is used, try a non-aldehyde fixative) to determine if autofluorescence can be reduced without sacrificing antigen detection. If aldehyde fixation is used and no other fixative can be used, then fixative-induced autofluorescence may be reduced by treating the sample with ice cold sodium borohydride (1 mg/mL) in PBS or TBS.

Another approach to reducing autofluorescence is to treat the tissue sample with dyes that quench fluorescence. These dyes include:

  1. Pontamine sky blue
  2. Sudan black
  3. Trypan blue

Paraffin-embedded samples are often more autofluorescenct, even though the sample has been thoroughly de-waxed. Under the circumstances, switching to frozen sections may reduce autofluorescence.

If these approaches are not sufficient to reduce autofluorescence while maintaining tissue sample detection, then the only other alternative is to just choose a fluorescent marker that will not compete with the autofluorescence. For example, fluorescence from dyes that emit at near-infrared wavelengths, such as Invitrogen Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750 and Alexa Fluor 790, are not affected by most tissue autofluorescence.


Additional notes on reducing high background staining
  • Carefully prepare tissue sample. Damage to the tissue can cause diffuse staining.
  • Prepare thinner sections if penetration of the detection reagents is insufficient.
  • Optimize fixation. Each tissue antigen will react differently with different fixatives. Optimize the pH, incubation time and temperature.
  • Blocking  may be improved by simply draining the excess buffer instead of washing the tissue sample prior to the addition of antibodies.
  • Use a monoclonal primary antibody instead of a polyclonal to reduce cross-reactivity.
  • Use cross-adsorbed polyclonal antibodies to reduce cross-reactivity.
  • Affinity-purify the antibody preparation on an immobilized antigen column. Many primary antibodies and almost all secondary antibodies are purified in this manner by their suppliers.
  • Decrease the concentrations of the primary and/or secondary antibodies to reduce nonspecific binding.
  • Decrease the incubation times with the primary and secondary antibodies to reduce nonspecific binding.
  • Choose an improved substrate that will produce a higher signal-to-noise ratio for the system such as metal-enhanced DAB rather than DAB, or kits based on TSA technology.

Additional notes on increasing staining intensity
  • Optimize fixation. The immunoreactivity can be affected by the fixative step along with the processing step. Avoid freeze/thaw cycles and high temperatures if the antigen is susceptible.
  • Use clean slides for mounting of tissue, and use appropriate conditions to prevent tissue from falling off the slides during processing.
  • Do not inhibit enzyme activity. If an AP system is being used, do not use phosphate buffer. If an HRP system is being used, do not use sodium azide. Both will inhibit the enzyme activity.
  • Do not over-block the tissue, since antigenic sites may be masked. In fact, excessive blocking can be worse than not enough or even no blocking.
  • Remember that neutralizing antibodies may be in the serum added to the blocking buffer.
  • Screen potential antibodies using a membrane-based system, such as dot-blots. If you use an ELISA to evaluate antibodies, bear in mind that protein conformations can be altered by adsorption to the surface of the wells in plastic 96-well plates. This means that it is possible that some monoclonal antibodies selected in an ELISA will not recognize native protein in the tissue.
  • Increase antibody penetration of the tissue by using unmasking agents such as trypsin, pepsin, chymotrypsin and Pronase. Additionally, try permeabilizing the sections with a buffered solution of Triton X-100 (0.1–1% (v/v) prior to staining.
  • Increase the detection efficiency, and possibly the sensitivity, by using signal amplification systems.
  • Properly prepare enzyme complex. Carefully mix all components of the enzyme–substrate complex in the correct proportions. Try to prepare dilutions of the enzyme conjugates right before use and don’t re-use them because enzyme activity is labile in dilute solutions. Never freeze diluted enzyme conjugates in an attempt to prolong their shelf life.
  • Avoid potential sources of biotin, such as nonfat dry milk or Fraction V-grade BSA (use only IHC-grade).
  • Increase incubation times or concentrations of the primary or secondary reagents.
  • Use a more sensitive substrate system such as a metal-enhanced DAB substrate.
  • Always run a positive control to determine if the system is working.
  • Use the correct counterstain and mountant. Some enzymatic products are soluble in alcohol, xylene or other solvents (e.g., aminoethyl carbazole (AEC)). Consider an aqueous mountant in such cases.

Recommended reading
  1. Beisker W et al. (1987) Cytometry  8:235–239.
  2. Cowen T et al. (1985) Histochemistry  82:205–208.
  3. Mosiman VL et al. (1997) Cytometry  30:151–156.
  4. Romijn, Herms J et al. (1999) J Histochem Cytochem  47:229–236.
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