I don’t know why I am doing this. I can’t handle it!” You thought you knew how to recognize and solve your IHC artifacts but after reading the troubleshooting guide the answers seem so far away. Long lists of tips. Examples too difficult to understand. Not-so-friendly images.

Feeling overwhelmed? You are not alone. Although sometimes troubleshooting seems unbearable, there is no need to worry! Troubleshooting is nothing more than the logical elimination of variables.

In our previous blog (Artifacts in IHC: the Usual Suspects - Part I) we have identified the major suspects (sample, antibodies, and protocol) responsible for the most common IHC artifacts such as lack of staining, nonspecific binding, and high background.

If you are now looking for tips on how to avoid artifacts in your IHC staining read on.

Overwhelmed by troubleshooting?

During the past few decades, improvements in the reagents and protocols used for immunohistochemistry have led to increased sensitivity of detection systems, contributing to the elimination of staining artifacts. However, it is still a daunting task to determine what is or is not nonspecific background staining and what is specific antigen staining or to identify the sources of background and the best approach to minimize noise and enhance the signal.

All that you need is a combination of the right elements, a dose of experience, a few tricks, and a grain of common sense. Take one thing at a time. Make a list of things you need to get done and start with it.

 

Tips to address a lack of staining

When the suspect is the sample:

Check protein expression in your tissue.
Insufficient penetration of detection reagents can be resolved by preparing thinner tissue sections.
Make sure the tissue is covered in liquid at all times.
Treat the tissue with antigen retrieval reagents to unmask the antigen.

When the suspect is the antibody:

Make sure the antibody works in your specific context (application + tissue)
Store antibody aliquots properly. Avoid repeated freeze-thaw cycles.
Use a secondary antibody that is reactive to the host species: if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody.

When the suspect is the protocol:

Adjust the fixation time or use a different fixative.
Check the datasheet to make sure the antibody has been validated in your specific application.
Increase the time of chromogenic detection
Increase or decrease the incubation time of the counterstain (since too much of the counterstain often obscures the specific staining).

Tips to reduce high background

When the suspect is the sample:

 Handle tissue specimens carefully during fixation to maintain their morphology: poor morphology makes the interpretation of the IHC staining inadequate no matter how good the antibody works.

When the suspect is the protocol:

If you use streptavidin as a detection system, the endogenous biotin must be blocked with an avidin-biotin blocking reagent before incubation with a primary antibody. This is particularly true for mitochondria-rich tissues like muscle and liver.

Preincubation with 5–10% normal serum from the species that the secondary Ab is derived from will prevent the non-specific binding of secondary Abs to endogenous FcRs.

Use 4% formaldehyde (10% buffered formalin for FFPE), acetone, or alcohol as fixative whenever possible: endogenous FcRs do not bind the Fc portion of IgG Ab after fixation in formaldehyde, acetone, or alcohol.

 

Tips to reduce nonspecific binding
 

When the suspect is the sample:

Always avoid drying out the samples.

When the suspect is the antibody

Always titrate your primary antibody. If the antibody works properly, the nonspecific staining should decrease as the dilution increases.

Use the secondary antibody obtained from another animal species rather than in the species of the target antigen. Make sure that it has been preabsorbed for the particular species you are interested in. Moreover, the immunoglobulin (Ig) isotype (IgG, IgM, etc) should match the type of the primary antibody. 

When the suspect is the protocol

Before adding the primary antibody, block with either 5% BSA for up to an hour with either BSA, serum from the same species as the secondary antibody species, or casein-blocking solution.

Washing steps between the staining, usually 3 washes with PBS or TBS for 5-10 mins each.

Use optimal antigen retrieval protocol for best specificity.

Move from RT to 4°C: cold temperatures slow down the reactions, increasing interactions specificity, and incubating overnight.

For fixed tissue, you can remove free aldehyde groups with 0.1% sodium borohydride in PBS for 30 mins prior to staining.

Reduce DAB incubation time. If you add hydrogen peroxide to your DAB solution, the color will develop slower and you can control the reaction more easily.

To prevent background caused by FcR incubate with preimmune serum or normal serum.

To reduce the endogenous enzyme activity, protocols that include Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP) may require reagents added to the chromogen/substrate to prevent non-specific signals, such as hydrogen peroxide and levamisole, respectively. Remember that hydrogen peroxide is unstable so make sure you make it fresh every time. As for AP remember that the AP isoforms in some tissues, like the placenta and GI tract, are resistant to levamisole and may require a heating step instead. 

Problem solved? prepare for future issues

Once everything is solved and fully functional, documenting the process becomes important. This is where you clearly document findings, actions, and outcomes. When the problem occurs again, there will be information available to walk someone through the means of troubleshooting and resolving the issue.

This documentation also captures a history of equipment and users so that perpetual issues become known and recorded. Remember that both positive and negative outcomes should be documented. This can save time during future troubleshooting and prevent others from making the same missteps you may have taken

Congratulations! As a crime-stopper, you are now ready to confidently report to your PI. Feel better already?

Readings

Ward J.M. and Rehg J.E. Veterinary Pathology, (2013) 51 (1) 88-101, Rodent Immunohistochemistry: Pitfalls and Troubleshooting

Buchwalow I. et al. Sci Rep. (2011) 1: 28. Non-specific binding of antibodies in immunohistochemistry: fallacies and facts

De Matos L.L., et al. Biomarker Insights (2010):5 9–20,  Immunohistochemistry as an Important Tool in Biomarkers Detection and clinical practice

Ramos-Vara J.A. and Miller M. A. Veterinary Pathology (2014) 51(1) 42-87, When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry: the red, brown, and blue technique.