Fixation on Histology

IHC Basics


IHC StainWe know that Immunohistochemistry is the combination of three distinct fields—histology/pathology, immunology, and histochemistry. With IHC we start with our properly prepared tissue samples (histology/pathology), take advantage of the fact that antibodies bind to specific proteins (immunology), and finally combine that with enzyme reactions that provide a colored end product to identify tissue components (histochemistry). The results provide diagnostic, prognostic, and even therapeutic information. The steps of the staining process are straightforward enough to be automated – and probably is in the majority of histology labs - but the process itself still must be understood. And a strong foundation of such basic understanding leads to ease in troubleshooting any unexpected staining results.

Just like with any staining procedure in the Histology lab, tissue must be prepared correctly in order to stain correctly. We can summarize the preanalytical steps:

  • Acquire sample
  • Fixation
  • Grossing
  • Processing
  • Cutting
  • Slide drying 

We know that other than processing, the above steps are not automated and therefore the possibility of result-altering error is present. For example, fixation time has only recently been recognized as so important to accurate IHC results that it must be recorded for some types of samples. And anyone that has tried to stain poorly grossed samples knows these can be hard to salvage. Documentation, Standard Operating Procedures, and training are crucial for instilling consistency with these steps.

Once pre-analytical steps have been completed, IHC staining can commence. These steps, in a very broad view, include:

  • Removing paraffin
  • Pretreatment
  • Blocking
  • Antibody incubation
  • Link incubation
  • Adding detection enzyme
  • Adding chromogen
  • Counterstaining

Of course, IHC staining is more complex than just this list. Whether pretreatment is required – and also what kind (enzyme digestion, chemical or heat-induced unmasking) - is usually a function of the target antigen and to some degree how the tissue was fixed. Similarly, whether blocking is required can depend on the detection enzyme/chromogen combination chosen (hydrogen peroxide or alkaline phosphatase), whether pretreatment steps may cause antibodies to bind non-specifically, and even the types of tissue being stained.

Then, of course, we must decide the best antibody to identify the target antigen: what are the advantages of monoclonal vs polyclonal? How to decide when an antibody cocktail is used for single or dual staining applications? What is the best way to stain antigens that are present only in very small amounts?

As stated previously, each step and choice can influence results. It’s impossible to cover all the consequences of those choices and multiple staining methods in a short blog post. I provided greater depth on this topic in a workshop I presented at the 46th Annual NSH Symposium and encourage those of you struggling with identifying the problems with unexpected staining results to brush up on IHC basics.

You can learn more about IHC Basics in the NSH Webinar: Basic IHC: Understanding the Process to Improve Your Results presented by:  Beth L. Roche,  HTL(ASCP)

1 comment



10-01-2021 13:29

Great overview!