Immunohistochemistry (IHC) Workflow


What is Immunohistochemistry or IHC?

Well, IHC is an immunoassay that examines tissue sections to identify distinctive protein (or other antigens), their localizations, and patterns. And traditionally, this is done by way of enzymatic labels and counterstains. So for this section of the discussion, we're going to talk about the enzymatic methods that correspond to IHC. And as we move towards the fluorescent methods or immunofluorescence, we'll talk a little bit about fluorescent methods for tissue immunoassays.


Types of Tissue

There are two types of tissue preparations for IHC. They are paraffin-embedded or formalin-fixed, which you usually see abbreviated as FFPE or frozen tissues. Now both of these tissue types have their advantages and disadvantages. And a researcher is going to choose them based on their needs, right. So the localization of their antigen, the location of their antigen, All of these are going to play a role in making their decision. But some basic advantages and disadvantages for FFPE tissues: They preserve the structure of the tissue a lot better. And they perform better for long-term storage at room temperature. The disadvantages: It's a very long procedure and fixation can mask the epitopes. Some advantages of the frozen tissues is it's a very quick procedure. It preserves enzymes and antigen function. But disadvantages, as you could imagine, if you're, you know, cryo-preserving tissue samples, ice crystals can impact the tissue structure. And it could be costly to store these at a low temperature for long periods of time.


IHC Workflow for Tissues

Let's continue by discussing the IHC workflow. Now, the workflow for IHC is very detailed and somewhat laborious. However, it could be broken down into the following broad steps. First, we start with preparation of the tissue, which are there be paraffin-embedded or frozen, followed by antigen retrieval, quench and blocking of the tissue, enzymatic antibody incubation and washes, or the staining step. Then followed by counterstain and mounting and then finally imaging with the brightfield microscope.


Tissue Preparation for Paraffin-Embedded, Fixed Tissue

We're going to begin with tissue preparation for paraffin-embedded tissue. But before we get into the details, let me just point out that there are probably slight variations to this example, depending on the labs preferences, their equipment, or needs. But this example is a pretty good depiction of the pe-tissue process. So to begin the first step in this process is fixation of the tissue with the fixative. There's numerous types of fixatives that you can use, but the most popular is probably formaldehyde or paraformaldehyde solution. And the addition of the fixative preserves the antigen of interest and its morphology, and it prevents necrosis of the tissue.

There are two different methods to fix the tissue. The first is perfusion, which is performed in a lot of small animal labs. In this fixation method, the fixative is directly injected into the vascular network of a small animal such as a mouse. Then the organ or tissue of interest is harvested and then dissected to the appropriate size. The other fixation method is immersion, where the sample tissue is harvested from the host. An example of this could be a human or animal biopsy and then literally immersed into the fixative solution. With the immersion method, you're relying on diffusion of the fixative into the tissue sample. The choice of type of fixation could be depended on many factors, and this can include the specific organ type being harvested, time constraints, the host of the tissue specimen, or even the availability of equipment for the methods.

Since this tissue prep method involves embedding your tissue in paraffin, the next step in the workflow is dehydration. And this step is needed because water, which is within the tissue sample, is admissible in paraffin. They do that mixed together, so you need to dehydrate the tissue before you can add it to the melted paraffin wax. And this is achieved by slowly increasing the percentage of alcohol as we see in this example from 70 to 90 to 100, and then into a non-polar solvent such as xylene.

After that point, the tissue should be pretty well dehydrated and you can move to embedding the tissue into molds and paraffin wax. And this is normally done with an instrument, a paraffin embedder. What it will do is heat the paraffin to a temperature where it is liquid. And after you remove the heat from the molten paraffin. As you can imagine, the wax will begin to harden. Therefore, embedding your tissue within the wax, which provides support when you're slicing your tissue samples.

Then step four in this procedure is cutting and mounting. The embedded tissue is cut with a piece of lab equipment called a microtome. And this also allows you to adjust the thickness of your cuts to your needs. And what you'll see is, as you cut, the samples are somewhat square in shape, and then each slices is attached to each other. So you could think of it where it almost looks like a ribbon of different tissue slices. And you place these slices into a warm water bath. And then when you're ready, you'll mount these slides. Or you mount these slices onto your slides. The slides that you use are typically coated with gelatin, and this helps enhance tissue adhesion to the slide. Then you take that slide and you sort of fish these tissue slices out from the water bath and place them on the slide. Then you dry them either by an air warmer or slide warmer.

And the final step in the PE tissue preparation process of the IHC workflow is de-paraffinization and rehydration of the tissue. And in this step, we need to remove any paraffin that may have penetrated into the tissue and also rehydrate the tissue to prevent poor staining. And this is done by going in the reverse order of when the tissue is dehydrated. Thus, you go from your xylene solvent to decreasing percentage of alcohol to buffers. And like I mentioned, this is a very, very broad overview. It's a very laborious method, but this is a nice summary of paraffin-embedded tissue preparation.


Tissue Preparation for Frozen Tissue

Now let's briefly discuss frozen tissue preparation. As you probably can tell by looking at the difference in this slide, this method is a little less tedious and there's no need for a dehydration and rehydration step here. But as you can see, the overall idea of frozen tissue prep is somewhat similar. Before we get in, just a brief note about fixation in frozen tissue sections, I should point out that if fixative is used in preparation of frozen tissues, the exposure time of that is much, much less than in PE sections. We'll talk about that in a bit more detail when we discuss antigen retrieval in the IHC workflow.

So in this figure, we can start with a fixation step either by perfusion or immersion, followed by cryopreservation. And cryopreservation is the freezing of your tissue to preserve it. And similar to paraffin, you need support for slicing tissue. So embedding is done in something called OCT compound using a mold and then frozen at -20 to -80.

Then the final steps are done in a piece of lab equipment called a cryostat, which allows you to slice frozen tissues while maintaining low cryogenic temperatures. Slice tissue sections are then mounted to appropriate slides and dried. And this particular protocol that I'm showing here on this slide, it shows cryopreservation before fixation. But you can also be cryopreserved prior to fixation if you're doing a fixative method. In the latter, fresh tissues are immediately snap-frozen in an isopentane dry ice mixture, OCT embedded and then sectioned with the cryostat and fix prior to staining. And again, the choice of frozen tissue prep method will be based on the researcher's needs and their resources.


Antigen Retrieval

Once your tissue samples are ready, you can move to the next step in the IHC workflow, which is antigen retrieval. And I want to talk a little bit about antigen retrieval and why it needs to be done on fixed tissue samples. First of all, most people do not antigen retrieve with their frozen tissue sections because the tissue has a difficult time holding up to the process without severe damage. The most popular choice for fixative of tissues is formaldehyde or paraformaldehyde, like we discussed on the previous slides. However, aldehydes have a tendency to mask antigens of interest as they typically result in cross-linking. So what crosslinking is in this case is the amino acid residues of one protein in the tissue sample are bonded to an amino acid residue of another protein.

And this is typically done by way of methylene bridges. And when that happens, the antigen can become buried. And therefore, you need to break those methylene bridges so you can retrieve this antigen. And the primary antibody can bind to it. So the primary antibody can bind to it, and we've tried to show that below in this figure. So the blue squiggle lines represent the proteins in the tissue sample, in the fixed tissue sample. And the red circle is the epitope on our antigen of interest. And as you can see here in this figure on the left, that the antigen is buried due to the crosslinking between the proteins and the tissues. So if a primary antibody were trying to bind to the antigen, it would be very difficult for that to happen. But if you break these methylene bridges, the antigen is exposed and the primary antibody could easily bind the epitope of the antigen.

And the two main ways that this is performed is either by protease-induced epitope retrieval, where a protease enzyme-like protease k, trypsin, or pepsin can be used to break the crosslink bridges or heat-induced epitope retrieval, which uses high heat to break the cross-links. In the heat-induced method anything from hot water bath to microwave and probably one of the more popular options is a pressure cooker, they can be used for this method. And again, the choice of method will be dependent on on the researchers needs and localization of the antigen as well.


Quench & Blocking

So now that we've talked about antigen retrieval, that's going to bring us to the next step in the IHC workflow, which is the quench and blocking steps. And these steps are performed to prevent nonspecific binding of different components, and that includes endogenous enzymes like peroxides and phosphatase or other proteins that are within the tissue and can react with any of our other detection reagents that we're going to be using for staining.

Now, to start, since the reagent used from the step onward are directly applied to the tissues on each individual slide, something called a pap pen is used to create a hydrophobic barrier around the tissue to keep the reagents specifically on the tissue area. So you take the pap pen, you draw around the tissue area. And in our figures below you can see that the tissue with the antigen of interest, which in our discussion is the red circle as well as other proteins. So you can see that on the, on the tissue. And after creating that barrier, the next step is to quench the tissue and we'll discuss the different detection methods very shortly. But remember, in traditional IHC an enzymatic detection reagent is used and either horseradish peroxidase or alkaline phosphatase are the enzymes of choice and both peroxidases and phosphatases are naturally occurring in tissue samples and tissues and therefore can react with the substrates that are used for detection.

So it's important for us to quench the tissues to prevent that reaction from taking place because it would provide, you know, nonspecific background. And HRP is the more used enzyme in IHC. So this example that we're showing here shows a quenching with hydrogen peroxide for using HRP. For quenching phosphatases a reagent called Levamisole can be used and after quenching and washing, the tissue is ready to now be blocked and the first block is performed with serum to prevent nonspecific binding of the antibodies used to the tissue.

So blocking reagents can include BSA and casein, but the preferred blocking reagent tends to be normal serum and more specifically, the species of serum chosen is the same as the host of the secondary antibody. So for example, if the host of the secondary antibody being used is goat, then the normal serum chosen would also be goat. The next two blocking steps are relevant when using amplification methods and IHC detection and again, we will talk about these shortly.

But most popularly an avidin/streptavidin approach is used for these amplification methods and since biotin is found naturally in tissues, it can react with the avidin/streptavidin used in IHC detection. So blocking endogenous biotin with avidin followed by blocking with biotin to block additional biotin binding sites on the avidin or streptavidin molecules is needed here. So overall these steps are very important to decrease nonspecific interactions and overall provide a very low background for the tissue.


IHC Worflow for Tissues

Before we move to the next step in the IHC protocol, let's just take stock of where we are in the workflow. We've talked about the laborious tissue preparation and the importance of the antigen retrieval and the two main methods to do so. And we discussed the importance of quenching and blocking a tissue sample. So that takes us to the last two steps, which are antibody incubation and washes was also known as staining and then finally counterstaining and mount.


Enzymatic Antibody Incubation & Washes

All right, so we finally made it to the antibody incubation or staining portion of the protocol. And similar to most other immunoassays, there are two main methods of detection, either direct or indirect. All IHC detection methods are going to utilize a primary antibody in the staining portion of this protocol. But whether or not the enzyme is conjugated to the primary antibody will determine if it's an indirect or direct method. If the enzyme is conjugated to the primary antibody, that is the direct method. And if the enzyme is conjugated to the secondary antibody, that is the indirect method.

And we see that depicted in the first two images below. Now sometimes the antigen of interest may not be present in high abundance in the tissue and this can be due to low endogenous expression. Or if a drug doesn't demonstrate good uptake to the tissue or perhaps even, you know, it's a wanted result of a knockdown in an experiment. In these instances, amplification methods can be used to increase the signal. Signal amplification methods build on the indirect detection methods. The two more popular methods exploit the high binding affinity between avidin and biotin. The two main methods are also known as the ABC method, or the LSAB method for biotin/streptavidin. In the ABC method, you begin with incubating the tissue with your primary antibody of interest.

The secondary antibody used is going to be specific for the primary antibody and it's biotinylated, meaning that it is, it has biotin conjugated to it. The secondary is then incubated with the tissue following the primary antibody incubation. And of course there's washes in between all these. And then a separate reaction, a biotin-related enzyme usually HRP, but could be AP, is incubated with free avidin molecules and the free avidin is derived from, this is a protein, and it's derived from egg whites of various bird species and reptiles and has the ability to bind multiple biotin molecules. It is actually tetravalent so combined up to four biotin molecules and these form large avidin biotin HRP enzyme complexes. So following this incubation, the separate incubation, a portion of this reaction can then be added to the tissue and any free biotin sites will bind the biotinylated secondary, resulting in increased enzyme concentration and thus increased or amplified signal.

So the LSAB method is somewhat similar and also uses a biotinylated secondary antibody. However in this method streptavidin, which is a type of avidin isolated from a species of streptomycin is is used instead of egg white derived avidin. But this interaction still has very high, very high affinity. The staining steps are also similar where the biotinylated secondary antibody is incubated with the primary antibody and the tissue. However, HRP or AP-conjugated streptavidin is incubated directly with the biotinylated secondary antibody, primary antibody tissue sample. Therefore all free biotin binding sites of the HRP-conjugated avidin can fill. And although both ABC and LSAB provide good signal amplification, LSAB seems to be the biotin method of choice as it has better sensitivity and it's smaller so it allows better tissue penetration.

The final amplification method is a biotin-free method, it is the polymer method, and this utilizes a long polymer with multiple HRP molecules conjugated to the backbone and this enzyme-conjugated polymer is conjugated directly to the secondary antibody. And this is a nice method because it allows for fewer steps and there's more enzymes present which would lead for more increased signal.


Stain & Counterstain

Now the IHC method is colorimetric and we do need to see the protein of interest in the tissue so we need to visualize that. But the HRP enzyme alone cannot do this, so we need a chromogenic substrate to fulfill this and the choice of the substrate that you would incubate with the HRP-conjugated molecules is key for the visualization.

And in the case of HRP, the substrate of choice is a chromogen called DAB. And when DAB reacts with HRP, it produces a brownish color which can be seen on the tissue and we can see this depicted below in the figure where we have the HRP-conjugated secondary antibody down to the primary. This is an example of an indirect detection and this Pacman-looking shape here represents the DAB.

So when this reaction takes place, you can see the brown color which identifies your protein of interest. So we see our IHC stain here. And I should point out that in both of these examples and the next one I'm about to go through, this is our cytochrome P450 primary antibody used in IHC as an example. And you know, just as a side note, if a researcher is using the AP enzyme, that substrate will be a chromogen called AEC.

Now the next step in this staining process is to counterstain and counterstaining is useful as it provides color to specific cellular structures, and this can help identify and better define the morphology. It also provides contrast to the substrate stain which is useful for determining localization of the protein of interest. Or it can also show distinct cellular compartments.

The most common counterstains that you're going to find in histology and the IHC method are hematoxylin and eosin and hematoxylin appears blue or sometimes purple, depending, and is specific for nucleic acids. And this is useful as it easily can identify the nuclei within the tissues. And you can see in both of these examples here, both on the stain and counterstain examples, the blue areas within the tissue that's from the hematoxylin. And what you're seeing there is the nuclei.

The other common stain used in histology is, is eosin and eosin is a dye that binds to proteins in the cytoplasm as well as connective tissue and also better defines the features of the tissue. And we see an example here of an eosin stain on the right in the counterstain example. And in most instances these two dyes are used together to give a full representation of the tissue structures and there's other different color dyes and types of dyes that you can use. And we'll talk about them later in the talk. But for the most part, these are the two most used dyes that you'll run into.


Advantages & Disadvantages

And that brings us to the end of our IHC workflow. But before we move on to our discussion about immunofluorescence, I do want to quickly point out that the majority of our detection methods are going to utilize indirect methods with a possible amplification step. I've laid out here some of the advantages and disadvantages of both methods, but you'll probably discover most people prefer indirect.