Working with a multi-step process that includes the fixation and processing of tissue, the mixing and selection of 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. It is important that training documentation is 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 quality of the staining should have a clear visualization of 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 working correctly on your positive and/or negative controls, one must first be able to recognize the problem and second, 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 automatic 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 cut down the technicians 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 on hand to help solve the problem. There is help available on-line from many sources, such as IHC world, the Histonet listserv, and the various manufacturers such as DAKO and Leica, and doing a basic search can find methods to a solution. To save time, it is better to develop a troubleshooting checklist that provides all the information for multiple problems that may occur in your laboratory.