Immunohistochemical methods: are they valid?

Note: This post is going to be greatly expanded at the moment I’m having trouble with my citation manager.

Immunohistochemistry (IHC) and immunocytochemistry (ICC) are examples of two immunohistochemical methods where a tissue sample is incubated with antibodies (Ab) to detect the presence of an antigen. The logic behind this method stems from immunology–that specific Abs bind to specific antigens e.g. proteins, chemokines, cytokines, receptors, membrane associated proteins, pathogens, etc. The pertinent difference between IHC and ICC is that in IHC you are working with cells that have been sliced open and in ICC you are working with whole cells which sometimes you permeabilize to allow Ab penetration into the cell.

The assumption behind IHC and ICC is that cells maintain their intra- and extra- cellular organization throughout the process of executing an IHC/ICC experiment and that antigens keep their position within the cell post fixation thus yielding valid data. Given that cells are composed of a mixture of proteins, lipids, and carbohydrates, as well as structured H2O and other complex molecules this is a troubling assumption.

[I doubt it’s an assumption in the true sense as most histologists and pathologists are aware that tissue morphology is altered during fixation. But the lack of attention to this issue in the literature and the amount of published data using immunohistochemical methods without confirming its validity leads me to call it an assumption.]

As a histologist you are trained in a variety of disciplines and you are trained to think about what is going on with the tissue you are processing at a low level as you expose it to different chemical agents. For some technologists techniques are developed and improved on by observation and trial and error. For other technologists a more evidenced based approach is taken where you consider the mechanics of a technique at the biochemical level to experimentally drive the techniques you are developing. The former is more likely to occur with diagnostic histotechs.

For example during fixation tissue samples can be dramatically altered. Most histologists should be familiar with the idea of fixation artifacts and the effects that different fixatives have on tissue morphology and that some fixatives can dramatically change the results of a special stain or IHC. The practice of antigen retrieval should be a clue to the chemical impact a fixative can have on cellular proteins.

Over the years there has been a consensus reached as to what fixatives are appropriate for various special stains and IHC/ICC. To say there is no logic behind this consensus would be misleading but to say that we know exactly how and why fixatives affect cells and overall morphology would also be misleading. Some of the science behind the choice of a fixative is evidence based but a good portion is observational and traditional and usually subjective.

The evidence behind that observation can be witnessed from the thousands of different histochemical techniques that histologists use to demonstrate structures within a tissue or cell. There is this phenomena that two laboratories can use the same histochemical technique and achieve different results. To compound that further two histologists can follow the same procedure and get different results. This is an anecdotal clue alluding to the fact that we really don’t have a precise and standardized approach when it comes to examining tissues using histochemical methods. There is some standardization and oversight, however, a lot of techniques are subjective. At the biochemical level thousands of variables come into play when developing an IHC/ICC e.g. purity and quality of reagents, the clone of the Ab, temperature, humidity, pH of reagents, etc. And the same is true with special stains.

While these things I’ve mentioned are the tip of the iceberg the real question is should we still rely on IHC/ICC in research and diagnostics to generate data? The answer is: it depends and requires consideration. I think to say immunohistochemical techniques are completely invalid would be an extreme overstatement. I also tend to think it depends on what type of antigen you are looking for. For example, is the antigen a chemokine or cytokine or is it a receptor or membrane bound protein.

In the future this area no doubt needs more research. We need more specific positive and negative controls and we need to be cautious in how we interpret results in IHC/ICC and in general any tissue that is processed with a histochemical method. When a histologist is trying to detect an antigen close attention should be paid to the nature of it and whether or not results can be affected by using different fixatives and the differences in cellular staining between whole cells in ICC and tissue sections in IHC where cells are cut open. For example, in ICC if you have intracellular perinuclear staining but in IHC you extracellular and non-specific staining using the same Ab one should indeed pause to think at a lower level. Questions such as: Is the fixative I’m using preserving the lipids of the cell or is the Tween 20 in my buffer causing a dissociation of the lipids from the cell structure?

There is no straightforward answer to any of these questions at this point. In the next post I will review some of the literature that addresses some of these concerns. At this point it is a unpopular topic so the list is short.

Edward’s modified Congo Red special stain


The Congo Red stain is a special stain used in histology to aid in the diagnosis of  amyloidosis. A polarizing filter will highlight amyloid an “green apple” color. This is modified version of the stain I developed for use in our laboratory:

Counterstain the slides in Harris hematoxylin for 15 minutes, dip in acid alcohol 1 to 2 times, rise in tap water. Blue in 0.3% ammonium hydroxide. Rinse in tap water. Incubate slides in Congo Red at room temperature for 30 to 60 minutes. Rinse in tap water, then dip the slides in 80% alkaline alcohol 1 to 2 dips. Dehydrate, clear, and coverslip.