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IHC Troubleshooting Guide

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

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Overview of Immunohistochemistry
(includes links to individual pages on all aspects and stages of IHC)

Vimentin detected by IHC Negative control of vimentin IHC
Immunohistochemical detection of vimentin in colon carcinoma tissue. Vimentin was detected in fixed human colon carcinoma tissue samples by incubating cells with an anti-vimentin antibody (left panel) or blocking buffer alone as a negative control (right panel). The samples were then incubated with Thermo Scientific Pierce Goat Anti-Mouse Poly-HRP, and the signal was developed with Thermo Scientific Pierce Metal Enhanced DAB. Cells were also counterstained with hematoxylin.

Strong Background Staining

The following points are provided to help identify the cause of high background staining, which results in a poor signal:noise ratio. Also see the Additional Notes sections at the bottom of this page for more information.

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Overview of Immunohistochemistry

Cause: Endogenous enzymes

Incubate a test tissue sample with 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.

  • Solution: Quench endogenous peroxidases with 3% H2O2 in methanol or water or use a commercial peroxidase suppressor. Endogenous phosphatases can be inhibited with levamisole.

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Blocking Endogenous Targets

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Peroxidase Suppressor

NBT/BCIP Substrate with Levamisole

Cause: Endogenous biotin or lectins

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

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 therefore won't bind to lectins.

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Blocking Endogenous Targets

Tech Tip #16: Block endogenous biotin

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Streptavidin Conjugates

NeutrAvidin Conjugates

Cause: Secondary antibody cross-reactivity or nonspecific binding

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

  • Solution: If normal serum from the same species of the secondary antibody is used to block the tissue, then increase the serum concentration by 2% or more. If blocking with another reagent (BSA, nonfat dry milk), then add 2% or more normal serum from the species of the secondary antibody. Alternatively, reduce the concentration of the biotinylated secondary antibody.

Egg whites are a source of avidin and are sometimes used to coat slides, dilute antibodies or block tissue samples.

  • Solution: Avoid using egg whites to prevent egg white-based avidin from binding biotinylated secondary antibody during IHC staining.

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Blocking Strategies for IHC

Immunodetection for IHC

Cause: Issues with the primary antibody

Nonspecific interactions between the primary antibody and nontarget epitopes on 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 concentration of the primary antibody.

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

  • Solution: Increase the blocking buffer 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--diluents usually contain 0.15M to 0.6M NaCl.

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Blocking Strategies for IHC

Immunodetection for IHC

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Primary Antibodies
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Weak Target Staining

Also see the Additional Notes section 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. Also, 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.
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Overview of Immunohistochemistry

Cause: Primary antibody potency

Primary antibodies lose affinity for the target epitope over time, either due to protein degradation or denaturation caused by long-term storage, changes in pH or harsh treatments (i.e., 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.

  • 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.
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Primary Antibodies
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Cause: Secondary antibody inhibition

While high concentrations of the secondary antibody can increase background staining, extremely high concentrations can cause 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 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 the diluent..
 

Autofluorescence

If a fluorescent marker is being used, check to make sure that there is no autofluorescence in the unprocessed, fixed tissue, which would suggest that the tissue preparation and labeling is causing autofluorescence. 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 causign the sample to autofluoresce. To determine if the fixation step is the cause of the autofluorescence, then 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 will work,then fixative-induced autofluorescence may be reduced by treating the sample with ice-cold sodium borohydride (1mg/mL) in PBS or TBS. (Beisker, et al., 1987)

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

  1. Pontamine sky blue (Cowan, et al., 1985)
  2. Sudan black (Romijn, et al., 1999)
  3. Trypan blue (Mosiman, et al., 1997)

Paraffin-embedded samples often give off more autofluorescence from the paraffin, even though the sample is cleared. Therefore, 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.

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Fixation Strategies and Formulations for IHC Staining

Immunofluorescence Detection for IHC

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Immunohistochemistry Reagents

Fluorescent Labeling Reagents

Additional Notes on Reducing High Background Staining

  • Carefully prepare tissue sample. Damage to the tissue can cause diffuse staining.
  • Prepare thinner sections if the tissue is not being penetrated well.
  • 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.
  • Decrease the incubation times with the primary and secondary antibodies to reduce nonspecific binding.
  • Choose an improved substrate that will produce a higher signal:noise ratio for the system such as Metal Enhanced DAB rather than DAB.

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Overview of Immunohistochemistry

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DAB Substrate

Metal-enhanced DAB Substrate

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 being removed during processing.
  • Do not inhibit enzyme. 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.
  • Remember that neutralizing antibodies may be in the blocking serum.
  • Screen monoclonal antibodies using a membrane system (i.e., a dot-blot). Polystyrene plates used to screen monoclonal antibodies alter the conformation of proteins bound to the surface. It is possible that 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.
  • Increase the detection efficiency, and possibly the sensitivity, by using signal amplification systems such as ABC.
  • Properly prepare enzyme complex. Carefully mix all components of the enzyme-substrate complex in the correct proportions.
  • 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 the 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. Consider aqueous mounting.

Learn more...

Overview of Immunohistochemistry
(includes links to individual pages on all aspects and stages of IHC)

 

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Immunohistochemistry Reagents

References

  1. Beisker, W.et al. (1987) Cytometry 8, 235-239.
  2. Cowen, T. et al. (1985) Histochemistry 82, 205-208.
  3. Mosiman, V. L. et al. (1997) Cytometry 30, 151-156.
  4. Romijn, Herms J. et al. (1999) J. Histochem. Cytochem 47, 229-236.
 
Written and/or reviewed by Jared Snider, Ph.D.

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