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Microscopy School Lesson 7 - Sample Preparation for Immunohistochemistry

Microscopic images are amazing; they allow us to see deep inside cells and tissues uncovering a world that otherwise would be hidden. Nevertheless, the outstanding image that represents the results clearly and with high quality relies on two key factors: 1) acquisition with the appropriate equipment and, 2) excellence in sample preparation. Neither of those factors can be disregarded because, if so, the final result will be deeply compromised.

Regardless of the equipment, sample preparation is a critical factor that is often disregarded by less experienced microscopists. In fact, irrespective of the quality of the imaging equipment, poor quality samples will always deliver a poor-quality image. Therefore, sample preparation is crucial for the overall imaging quality.

There are several steps for Immunohistochemistry sample preparation. It begins with adequate sample collection and fixation. Subsequently, the samples are stained with antibodies. Antibody staining is crucial and dependent on several factors that must be determined experimentally.

In recent years together with the increasing microscopy power to image large samples, several post-treatment clearing techniques have emerged, allowing in toto imaging of fluorescently labelled samples. Currently, all these new technological advances enable the imaging of biological samples with an unprecedented morphological contextualization.

In this webinar, Dr Marco Campinho (CBMR - Universidade do Algarve) discusses every aspect of sample preparation for Immunohistochemistry.

Learning Objectives:

  • List the critical steps in sample preparation for Immunohistochemistry. 
  • Recognize the caveats in sample preparation.
  • Design and plan an immunohistochemistry experiment.

Questions Answered: 

  • What are the critical steps in Immunohistochemistry sample preparation? 
  • How to optimize antibody staining? 
  • What is clearing?

Hello, everybody, my name is Marco Campinho, and I'm here to give you an overview of immunohistochemistry sample preparation. Sample preparation is the most determining factor in achieving high-quality results, you can have very sophisticated and outstanding microscopy in the equipment and hardware, but if your sample preparation steps are not the most adequate, you never get the high-quality results that you want to achieve. For this, customization of each experiment is key, because it involves a huge amount of different steps and a huge amount of different processes that you have to carry out in order to enable the labelling and detection of your target of interest. And of course, as any other good experiment, it starts with a good experimental setup. As I said, in the beginning here, we are going to focus on immunohistochemistry of fixed samples. Immunohistochemistry takes into account eight key steps that are involved in any immunohistochemistry preparation. It starts with sample collection, fixation, pre-treatment, blocking, incubation with your antibodies of interest washing, detection, and specimens preparation for imaging.

In sample collection, it is very important to be careful and fast in the manipulations that you are carrying out. This is especially important when you are dissecting tissues from animals because the process is that you are interested in observing and studying might be prone to changes if you take too long to collect the tissues. As well, anesthetics that you not only have to use when you're dissecting the tissues from animals can have a huge impact in the biological phenomenon that you want to study because they will change the physiological condition of the animal. And given that the dynamics of the phenomenon can change dramatically the results that you are going to achieve. As well, the use of the adequate vessels and supports to collect samples is important.

So the fixation which is key for immunohistochemistry starts with selecting your fixative adequately. Two key points should be in your mind all the time. First, antigen preservation and also tissue and cell integrity. And this is because both the fixation conditions can change both the antigen and, as well, the cell integrity and morphology of your issue. The most widely used fixatives are cross-linking fixatives. These are most the most common, formaldehyde and glutaraldehyde. They fix proteins by cross-linking them to each other.

Nonetheless, formaldehyde, for instance, reduces tissue size, whereas glutaraldehyde leaves three active aldehyde groups in the tissue that tends to give non-specific fluorescence for that particular tissue. You can also use coagulants like ethanol and methanol, they're throughout within fixing proteins to each other by covalent links they coagulate or precipitate the protein with each other in making them stiff and very close together. They do not fix carbohydrates and lipids. And they have the advantage of not introducing fluorescence and reduce the usage of detergents because they themselves promote permeabilization of your cell.

Another option is acids like acetic acid or picric acid. They also precipitate proteins and do not fix carbohydrates and lipids. As a last option, you can always use cryopreservation. It's actually the best for conserving tissue, cellular and epitope integrity. However, before fixation, they need to be buffered with osmolites, normally sucrose, in order to prevent the formation of ice crystals inside the cells that will disrupt the tissues and disrupt the cells and promoting the leakage of your targets to the outside and to places where they are not supposed to be. And as I said before, this is a fixation step that before requires some processing. As a rule of thumb, I always use a fixative volume that should be at least 10 times higher than the mass of the tissue and this is because especially of adequate penetrability of your fixative into the tissue. If you have a big volume of fixatives, this will promote a more homogeneous fixation and will allow you that even bigger tissues get uniformly fixed and preserve their integrity.

Another aspect that you should always bear in mind is that you should always buffer your fixative because this will prevent any osmotic shocks that might occur during the fixation process as well. Time and temperature of fixation is important because we are promoting chemical reaction or physical chemical changes in your sample and both time and temperature will influence those. So, a good equilibrium between time and temperature will allow you to have the best results possible. Another aspect that you have to think about is if you want to fix by immersion, this being putting your tissue into a vessel with the fixative and that the fixative will then permeate through the tissue and fixate it. Or, for instance, if you want to fix organs in an adult animal like mice or another zebrafish, you should think about perfusion. In perfusion, you change the blood by the fixative, and the fixative will get into the animal to the tissue through the blood vessels enabling a very in-out fixation allowing for very uniform fixation and this is especially important in very big samples.

After fixation, it's really important for you to wash extensively in order to remove the fixative. Most fixatives are highly reactive chemicals that if you don't remove them properly, they will also react with the reagents that you're going to use next, especially antibodies which are also proteins. And because most fixatives target proteins, if you don't remove up all of them that are there, you risk that you damage those reagents and will not have the results that you are expecting. It's also very good to use detergents during the washings after fixation because they will promote a higher penetration of the washing solution and allowing a better removal of all the reagents inside.

And then after washing, long term storage is possible, and this normally is carried out at minus 20 in methanol and methanol is very good advantages. First, it will allow the removal of some lipids from the cell membranes promoting a better access of chemicals later on. And because it doesn't freeze at minus 20, it allows you to keep your samples in very stable conditions that you can use later on when the time is more appropriate. However, fixation is this combination of ideal versus practice. Ideally, a good fixation method should preserve the tissue integrity as a whole. In practice, the chemical fixative is usually selective. For instance, like formaldehyde, it will act on the other aldehyde groups of proteins. So, in this way, you should always choose a fixative for the molecule of your interest, or use a combination of fixatives, or use a physical fixation method like rapid freezing and cryopreservation.

Ideally, as well, a good fixation method should preserve the cell structure with minimum changes from the living state like volume, morphology, localization of your target in the subcellular spaces where it should be. However, in practice, fixation in tissue processing usually induces artifacts that they change the volume, they change the morphology. So work in order to minimize these avoidable ones. And this could be achieved by decreasing the temperature or diminishing the time of fixation. But in the end, you always have to interpret the tissue structure in the context of the fixation in the process.

After fixation, some pre-treatments might be necessary, either for the removal of pigments that will interfere with your imaging that you have to carry out later on. For instance, melanin can be very detrimental in microscopy because it prevents light to go in and out of your tissue, thus masking and creating artifacts in the imaging process. Or you have to permeabilize with different pre-treatments in order to allow your reagents, namely the antibodies, to get into their targets. And you have different approaches like detergents of polar solvent washes or enzymatic treatments. And in many cases, because it's not possible using any fixative technique to preserve the epitope that you want to identify, you have to retrieve it.

So permeabilization pre-treatments, it's not only used with detergents, there are two main classes of detergents, denaturing detergents like Tween-20or non-denaturing detergents like TritonX. The apolar solvent washes are also sometimes necessary like, for instance, treatment with acetone in order to allow the removal of some components of cell membranes and allow the creation of holes in the cells and in the tissues that allow better permeation of your regions to detect your target of interest or enzymatic treatments. And you can use trypsin, it will be more targeted, but you can also use less directed enzymes like protenaise K that will attract every single protein, but you have to bear in mind that these type of treatments with protenaise K can give you huge amount of artifacts, can destroy your tissue and you should always be careful and monitor very attentively your sample. Last, Collagenase is also a very good option because most animal tissues have collagen-rich extracellular matrix that is very impermeable and treating with collagen permits a very directed degradation of Collagenase and the introduction of higher permeabilization capability in your sample.

Epitope retrieval is also sometimes necessary because the way by which the chemical aspect of fixation goes about, it masks the three-dimensional conformation of your epitope and you have to retrieve it or else your antibodies are not going to be able to recognize it. For that, you can use chemical retrieval like using acids, ketones, or urea, or, which is very widely used, heat-induced retrieval either by radiating your sample with microwaves or immersion of your sample in a boiling solution of citric acid for a given amount of time. Of course, any of these types of heat-induction retrieval are dependent on the time you take for them. Too little might not do anything to your epitope, too much could also induce a different confirmation of your epitope that becomes as well impossible to detect. So we really have to find the sweet spot of time of this type of treatment in order to have the best result possible.

Nonetheless, I'd like to just give some important notes about pre-treatments, that they will always change the biological results because they are, in general, very harsh treatments to the tissues and they can induce non-specific background signal as well. So this should be one of these aspects of immunohistochemistry preparation where experimentation and careful changing the conditions and careful observation of your tissue or cells should be in your mind all the time.

After this, we normally go to blocking. And in blocking, our aim is to reduce the non-specific interactions of antibodies with tissue proteins other than the target. Antibodies are very large peptides that they have a huge amount of surfaces that can interact by electrostatic interactions that are not specific with a huge range of different proteins in your samples. And what we want to achieve with blocking is to reduce as much as we can these interactions. Common blockings is bovine serum albumin, BSA, dried milk, animal serum, or you can also use antibody saturation, and in this case, what you do is that you use a highly concentrated solution of your antibody that you pre-incubate with a tissue of interest that is not going to be used for your analysis in order to allow antibodies that interact non-specifically to be attached to that tissue. This is especially important in polyclonal serums because they have a higher diversity of antibodies that are able to detect your target of interest and might have antibody species that are not as specific as you would wish for.

After blocking, you have the incubation with the antibodies in itself. And for this, we rely on the selectivity of antibodies, their capacity to give us the resolution that we need to pinpoint a single molecule in the middle of a hugely heterogeneous sample. And as well the capacity to label multiple targets in the single specimen, which is quite useful if you want to see interactions and conversations. So, for this incubation, you first use the primary antibody and this primary antibody is the one that actually detects and interacts with your target in your sample. This can be monoclonal, this meaning that they are only of one species of antibodies, they only detect the same epitope or polyclonal, which are normally produced and generated after the injection of an animal with your antigen. After that, the animals are bled and the antibodies retrieved. And here you produce a collection of antibodies that have the capacity to bind your antigen at different locations, thus allowing you to have more than one region of your target to be viable. This can be very advantageous if you are trying to target something that is very scarce in your tissue and can increase your capacity to detect it.

After using the primary antibody, we will use a secondary antibody that will detect the primary antibody. This can be species-specific, which is very advantage because you might want to detect one target with a primary antibody made in mice and the second target with an antibody made in rabbit. And by then using secondary antibodies that only detect either mice or rabbit, you can have the labelling of both primary antibodies in the same sample and detect both targets in the same sample. As well, if you do not have the possibility to detect both targets with secondary antibodies made in different species, you can actually still have the chance to use secondary antibodies that are able to discriminate between different classes of antibodies. For instance, if you're only having mouse antibodies for both targets that you want to detect in your sample, when one is an IgG and the other one is IgM, you can use secondary antibodies against mouse IgG and mouse IgM and still be able to detect both targets at the same time.

As well, if your primary antibodies are not class-specific and are both from the same class but that from different isotypes, you can also have at your disposal secondary antibodies that are able to discriminate between different isotypes of, for instance, IgMs and you can detect an IgM1 and an IgM2 from mice and be able to label specifically and without interference from one another the two targets using the first antibodies of mice. As well, you can then have the choice to use conjugated with either fluorochromes or enzymes. And this will allow you to use one type of technology in microscopy, or another type of technology in microscopy, they both have advantages and disadvantages, and you can then have that further choice.

In general, while you do the incubation with your antibodies, low incubation temperatures tend to favor specificity because they will create more stringent conditions for the antibodies that will allow or that will favor that the antibodies will bind specifically to your target and or have less non-specific interactions with the other proteins in your tissue or sample. Of course, agitation enables you better results, because it enables a better homogenization of the solutions and of your antibodies into the tissue. And you should always bear in mind that if you are doing immunohistochemistry in slides, evaporation and drying of the samples, it's your worst enemy and you should always take care to avoid these type of issues all the time, like, for instance, using wet chambers during the incubation time.

After washing, you are using very highly concentration solution of antibodies. So this means that even after you taking out the antibody solution, you are going to have their antibodies that are just stuck to other proteins that are not your target. So you have to wash extensively in order to remove all of these. As a general rule of thumb, I always use at least five times greater volumes of washing solution than the mass of the tissue that I want to wash. This will allow too for a greater dilution of the antibodies that are non-specifically bound and it will enhance your capacity to release those non-specifically bound antibodies from your specimen and our tissue. Room temperature is normally desirable because it increases the entropy of the solutions and increases the stringency of your washing conditions. And by increasing these washing conditions, antibodies that are not tightly bound like the ones that are not bound to your target will get released from tissue more easily and allow you to have a much cleaner sample.

And I always tend to over-wash than under-wash. What do I mean with that? I prefer to wash several for longer times and more often. Although it takes me more time to run the procedures, in my experience, this is revealed that you have less background and you have more precise signal in the end of your experiments. As well, it's highly desirable that while doing the washes, you use detergents. I normally tend to use TritonX, which is a non-denaturing reagent, so that not to allow the solution also to disrupt the specific binding of my primary antibodies or secondary antibodies with their targets. Or if you have a more, kind of, dirty serums, Tween-20, which is a denaturing detergent, can be quite useful in taking away those more recalcitrant serums.

After washing comes the detection, which you can have two methodologies, can be enzymatic where you promote the deposition of a chromogenic dye into the region where your antibodies are bound, or fluorescence where you use a fluorochrome that is bound to your secondary antibody. So, during enzymatic detection, you have two enzymes that are normally conjugated with a secondary antibody or with a complex of avidin and biotin, which are alkalyne phosphatase and horseradish peroxidase. These enzymes, by conjugating with avidin and biotin, you can increase the number of enzymes spurred antibody where they are bound. And as you can see here in this cartoon, you add the primary antibody here in step one, and then you add the biotinylated secondary antibody. Afterwards, you add the avidin by attenuated enzyme complex and you add the enzyme-substrate, thus allowing that enzymatic reaction turns the enzymatic substrate from soluble to insoluble and that creates a deposit of that insoluble chromogene in the region you're aware your antibodies are bound.

And this is the type of results that you can achieve. Here, you can see that one detection is done in blue and another detection is done in brown. And you can actually have a huge amount of combinations in different colors according to the types of enzymes that you use and the type of dyes or substrates that you can use. And it can be very versatile. Normally, florescence is more widely used because it enables the usage of different microscopy technologies like confocal, lightsheet microscopy, and it's more practical in many ways because it involves less steps. Basically, your primary antibody binds to your protein of interest. And then the secondary antibody which is already bound with fluorochrome binds to the primary antibody and one molecule stains one molecule.

Fluorescence as well has a huge amount of advantages. Because of the existence of different fluorochromes, you can exploit all the capacity of detecting multiple targets in the sample by using different fluorochromes. And because of the existence of so many, you have the entire spectrum of light in order to have different combinations and you can have three, four, or even five different targets labelled in the same specimen. So that's very advantageous. And as you can see here, this gives you a great amount of advantages because you can detect a single cell in a single tissue with very high specificity, as you can see here in the left, or you can detect one cell very specifically in a whole heart of zebrafish, for instance, as you see here in the right.

However, when detection is carried out, the immunohistochemistry procedure is not over yet, you still have to prepare your specimen for imaging and you have to bear in mind three key points. You have to gradually equilibrate your specimen with the embedding media, you have to fit the medium to your experiment, and you have to have different mediums have different characteristics. So you have to gradually equilibrate your specimen will the embedding median in order to preserve the morphology, and also to have a very homogeneous sample that when you are imaging you don't create artifacts just because one part of the sample is less transparent than the other.

As well, you have to fit the medium to your experiment because maybe you want to do a permanent or non-permanent preparation or because you are using a chromogenic or fluorescent labeling. And that is important because different embedding medias can react with either the chromogenic or the fluorescent labeling that you're using, and can either decrease or create artifacts in your imaging that are simply not there. And of course, you have to think about if you're using a thin or a thick sample. And the embedding media could actually be your good ally in order to create more transparency or less transparency.

As well, one of the things that you have to bear in mind with the medium that you want to use is if the objectives that you will have available in your microscopy facility and the microscope that you want to use are adequate for that medium. Because if the mediums are not adequate for the optical properties or your objective, you are always going to have subpar imaging and you're always going to introduce artifacts in your images. As well, as I said before different medias have different characteristics, they change morphology, and this is a very important aspect because this happens almost all the time. And they can have higher or lower transparency.

And here I am showing you some experiments that I carried out with some sole, which are labelled by calcium in green, that was calcium deposits either in bone or, for instance, in the eyes, and also immunohistochemistry of labelling of muscle tissue, which you see in red. In glycerol, glycerol, it increases the transparency of your sample quite a lot, but not as much as we desire, especially in these thick samples of 1-millimeter thick. So we started to try out some other reagents like scale A. And what we found that scale A, as time passes, increases the transparency of the sample considerably. But as the incubation time in these mediums is carried out at six days of incubation, for instance, you can start seeing great changes in the morphology of the animal that just it's...artifact that is not desirable. And this was better achieved at three days than six days. However, you also notice that while the immunohistochemistry of labelling of muscle is enhanced by scale incubation time, the signal of calcium is greatly reduced, as you can see in the eyes and in the skull. The calcium signal, it's decreased significantly as time goes by.

Something that I also wanted to talk about is some new emerging technologies that are now coming to fruition for the imaging and preparation of sample by immunohistochemistry. One of these emerging technologies is expansion microscopy. So expansion microscopy is four fundamental steps. You stain, and after staining of your specimen, you link it to a gel matrix that then goes by a digestion process where it's broken apart, and then it's expanded. And this is very useful because it allows you to image small structures using more conventional microscopy techniques and allows the researchers to identify small structures that if they were not expanded, that would be difficult. It uses a polymer system to do this and it's able to expand the sample size up until 16 times its initial size and can be very useful in certain situations.

A new emergent technology that I think it's going to be quite promising is fixation with polyfunctional crosslinkers. This is a technology that it's still quite expensive. But this fixation method preserves the protein conformation of your sample, so much so that it preserves the endogenous fluorescence of gene-encoded fluoroflors like GFP and ISH. It's quite advantage. It's one less incubation that you have to carry out and it allows for you to perform both immunohistochemistry of other targets and also in situ hybridization of genes of interest with high spatial resolution. And I am sure that you will see more work coming out in the near future with these type of fixative reagents, which I think it's very promising.

Finally, as a take-home message, I'd like to just review these three notes that a good image starts always with adequate sampling and fixation. Where you have to optimize your antibody conditions all the time because fixation changes, your species might be different from the ones where the antibody was first generated for and you have to optimize for that particular conditions, your sample dimensions might interact with the way by which your antibody detects your target. And post-processing of samples is determined by specimen size and verbal microscopy. So you have to also bear in mind that for you to have the best imaging possible, you have to have the most adequate impeding media possible.

As a final note, I would just like to say that I always have this mantra for my students, it's better to repeat an experiment, than use image processing to achieve the quality results that you need.

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