Technically, immunohistochemistry is relatively simple and straightforward. However, there are a lot of variables that must be identified and optimized for each study. Immunohistochemistry may also involve troubleshooting of a variety of factors.
The right antibodies and controls
I am about doing things right the first time. A simple, but sometimes overlooked step is to choose antibodies that work for immunohistochemistry. This can save you a lot of headaches down the road. Search for specific antibodies from literature, vendors, as well as your peers. There are several things to keep in mind with antibodies. What might work well in one laboratory might not be optimal for your laboratory. Each antibody needs to be tested with your staining system. Antibodies overtime can lose their staining intensity. Exposure to air and light can cause this.
Sometimes we overlook the importance of our controls. Date and number each batch of controls that you cut. Keep track of who cut what and when. Tissue sections kept at room temperature for more than a few weeks show loss of some epitopes. Some antibodies are more sensitive to this than others.
When we get into troubleshooting, it is important to set objectives to determine a goal for each run. Change one variable at a time such as incubation time of primary or detection system or the pretreatment method used. It is also very important to document your results. If optimization is not successful or if problems with optimized antibodies arise, troubleshooting may be necessary to resolve the staining issues.
Weak or no staining
Two main problems are common with IHC staining: Weak or no staining as well as non-specific staining and/or background.
One of the first things is to check with weak or no staining is the primary antibody. It may have been omitted by mistake. This is especially true if you are making dilutions. It also may be defective or out of date. Check the expiration date of the antibody. It also may be necessary to use a new lot or batch. Antibodies lose potency over time and may require higher concentrations. As we get busy in laboratories, mistakes can happen. Sometimes we need to recheck our calculations to make sure that we did our dilution correctly. Do not overlook the importance of the diluents that we use. Some may contain substances that interfere with staining. Sometimes we might be dealing with inactivation of the antibody. I found this to happen most often when I had freeze-thaw patterns with the antibody. This typically happens if we freeze small aliquots of certain antibodies. Wrong incubation times or temperatures may also need to be addressed.
Antigen retrieval can also play a role with weak or no staining. The method that we have chosen may be inappropriate. Some of our instruments allow us to increase our times in the retrieval. You can also look at the other retrievals that are available to you. Also keep in mind that some antibodies do not require antigen retrieval. Check pH before use and also ensure that our concentration calculations are correct. We need to be sure that our temperatures and incubation times for our retrieval are correct. Do not rule out the opportunity for dual retrieval for difficult antibodies. Heat and enzyme retrieval can work well together. Start out with a ratio of ten minutes in heat and five minutes in enzyme or vice versa. Tenascin C is an antibody that comes to mind for this. If all else fails, refer to the specification sheet for the antibody you are working with.
Not using the appropriate control can play a role in weak or no staining. Sometimes when you purchase control slides, animal tissue may be used. You may also have a control that has low expression level. Keep in mind that you may have cut past the area of interest. Take charge of your controls and start looking for drop off before this becomes a major problem.
Ancillary reagents also play a role in weak or no staining. Incorrect order of steps can be a problem. Distilled water pH is a problem that I encounter in the of our water. Distilled water can contain inhibitors of peroxidase reaction. Again, some antibodies are more sensitive to others with this phenomenon. Also when you troubleshoot the water, do not forget the buffer. Distilled water is often times used to dilute the buffer for your final working solution. The buffer may also be contaminated or out of date. Also check your detection kit for the same reasons.
As we continue to discuss weak or no staining issues, we need to turn to the protocol itself. Incubation times in our protocols can be too short. This also applies to our retrieval protocols. We are taught that washing is a very good thing. Keep in mind that our washes need to be stringent but brief. You can cause dissociation of antigen-antibody complexes by too extensive or vigorous washing. Also watch the washes with red detection kits. We can completely wipe out our staining or cause the bleeding that we sometimes see. When we look at our protocols, we have to think outside the box. The peroxidase block can inhibit staining based on where it is in the protocol. Moving the peroxidase step down in the protocol can enhance staining. PAX5 tends to work better when the peroxidase block is moved from the beginning of the protocol to after the polymer step. Too many blocking steps can also inhibit staining.
Mounting media is another variable that should not be overlooked with weak or no staining. Choose the correct mounting media for the utility that you are using. This is also something to consider when working with red detection kits. Some enzymatic products are soluble in alcohol, xylene or other solvents.
Over-staining and background staining
Over-staining is another common problem that we see. Again, we need to start with the protocol itself. Incubation times of our protocols, as well as antigen retrieval, may need to be shortened. If you are working with concentrates, a dilution study may be needed to determine the correct concentration for your primary antibody. Sometimes the incubation temperature for your primary antibodies or antigen retrieval is too high. The substrate incubation times may also need to be adjusted.
The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. These hydrophobic interactions can be decreased by formulation of the diluent buffer. This can decrease the buffers ionic strength. Tween 20 detergents as well as a 1% bovine serum albumin can also be added to the diluent to reduce this non-specific binding.
When we experience non-specific or background staining, the blocking step must be considered. Ensure blocking solution is aligned with species of antibody used. Increase the concentration of our blocking solution until the desire results are achieved. Increase incubation times as necessary. High background can also be caused by too strong of concentration of your primary antibody. Non-specific binding can also arise from our secondary antibodies. You can also experience problems when you choose an antibody that is made in the same species as the animal that you are testing.
We also need to review our antigen retrieval when we experience high background or non-specific staining. Over retrieval is often the problem. This can be adjusted by decreasing our retrieval times or temperatures. We can also choose a lower pH retrieval.
Over digestion can also cause this problem. Look at your enzyme retrieval time, temperature and concentration.
The protocol may also be adjusted for non-specific or background staining. We mentioned the pitfalls with over-washing. The converse is also true here. If we do not wash adequately, some of these issues may occur. Do not let your slides dry out!
Sometimes it can be very frustrating when tissues come off of our slides. One of the causes is inadequate drying of the slides after cutting. Also be mindful of too aggressive of antigen retrieval. Do not forget enzyme retrieval here. Do not rule out a bad lot of charged slides. I have experienced this myself.
Brown/black pigment is something that we sometimes see on our slides. Make sure that proper maintenance is done on our instruments. Also check the DAB itself. Sometimes we may also see this from the polymer step in our detection protocols. I have also seen this from a lead pencil when writing on the tops of the slides.
Hopefully these tips will help you out. I know there seems to be a lot that can go wrong, but with a little perseverance, you will get a nice result. Make sure that you find a protocol that is right for your laboratory and remember, troubleshooting is everyone’s job.