Working with a multi-step process that includes the fixation and processing of tissue, the mixing and selecting appropriate reagents, and obtaining interpretable results requires proper training and education of all personnel performing IHC. Any one of the steps in that multi-step process can cause false-positive or false-negative results. Training documentation must be in place for those who perform the IHC staining. This is written verification that training occurred, and each was signed off as competent to perform these highly complex procedures.
The staining quality should visualize the stained area with little or no background staining to interfere with interpretation. When things go wrong, it is often difficult to determine the cause. When a stain does not work correctly on your positive and/or negative controls, you must first recognize the problem and then know how to initiate a troubleshooting method. The most common and best method is to add one IHC reagent at a time to determine at which stage/step the problem may have occurred. This is very time-consuming but necessary.
Many manufacturers of automated stainers have provided charts that they have developed for troubleshooting specific scenarios, and most IHC reference books have a paragraph or chapter on troubleshooting. All publications are helpful and usually reduce the technician's time searching for the answer. It is good to have one good reference book and a good chart from one of the automatic stainer manufacturers to help solve the problem.
