Posted in | Immunology | Lab Techniques

An Introduction to Immunohistochemistry Principles with Troubleshooting Tips Webinar

Watch this on-demand webinar presented by Dr Carolyn Miazga.

Discuss common IHC problems and review tips and techniques for your experiments.

About the Presenter

Dr Carolyn Miazga received a BS in Biology from Boston College in 2003 and a PhD in Biology from Tufts University in 2009. Her dissertation focused on embryonic heart formation using the model system, X. laevis.

Carolyn's IHC experience includes using whole-mount and tissue sections as samples and troubleshooting for both techniques.

Webinar Topics

  • Sample processing
  • Antigen retrieval
  • Signal amplification
  • Controls
  • Troubleshooting
  • Antibody selection

Webinar Transcript

Hi, and welcome to Abcam's first IHC webinar. Today, I'll be discussing immunohistochemistry, so those of you who are not familiar with this technique, I'm going to go over what it is, how to perform this application and then finally some common problems that researchers might encounter, and how to solve them. So first off, what is immunohistochemistry? Well, immunohistochemistry is a technique used to study microscopic anatomy. Tissues are sliced into very thin sections and then are mounted to a slide, they can be then observed under the microscope. Here we're looking on the left at a mouse liver tumor with pan-cytokeratin staining in brown. As you can see, you can easily see the distinction; very distinct stain and localized to the area of protein expression. So this technique allows researchers to study protein expression and localization, and it's widely used in basic and clinical research.

​So how does immunohistochemistry come about? Well, I see it as a variation of histochemistry; this is a technique used by histologists to examine tissue morphology. With histochemistry the stains are much more general, so here the common stain used is hematoxylin and eosin. Hematoxylin will non-specifically stain a nuclei a blue color, and the eosin will counterstain the cytoplasmic regions with a pink or reddish stain. Well, this is a great technique to observe tissue structure and abnormalities, the stain does not distinguish among proteins, so there's no specific information about what's going on in the tissue.

​In order to get around this issue, immunohistochemistry was developed and using specific antibodies IHC can distinguish among proteins. So here we're looking at a tumor biopsy section and this is the bone marrow, and here in the brown you see a stain for the protein Wnt1 and, of course, this is a hematoxylin counterstain for the nuclei. So, clearly, you can see that the protein is fluoresced in portions of the tissue, but not in the whole tissue, so you get this local information that you don't get from histochemistry.

​So now I'd like to go into how immunohistochemistry is performed. I'm going to divide this into two main elements: the first is sample preparation. This will involve a fixation of the tissue, embedding into the matrix so that it can be sectioned, and then finally antigen retrieval. The next element that I'll discuss is the actual IHC protocol. I'm going to go into blocking: what it is and why it's important? Incubation with a primary antibody that will target your protein of interest. Incubation with a secondary antibody which will be specific for the primary antibody, and this allows, ultimately, for your detection. Then I'll go through adding an enzyme substrate, so for enzyme conjugated secondaries this is going to be a very important step and then, finally, coverslipping and observation.

So let's first begin with the sample preparation. Collectively, these steps are referred to as histotechnique. This includes fixation, embedding and sectioning. Now I'll begin with fixation. So the point of fixation is to preserve post-mortem on the structure and composition of a living tissue, and there are two main approaches. One can use a cross-linking reagent, such as paraformaldehyde or an organic solvent such as acetone, methanol or ethanol. First, I would like to describe the cross-linking-based fixative, and these involve the use of formaldehyde or some variation of it. The two most common fixatives are formalin, which is an aqueous solution of formaldehyde and water, and this is prepared as a 10% solution. The one disadvantage to this type of fixation is that as the solution ages, methanol is formed as a by-product and this can result in a clumping of the proteins instead of cross-linking. This will present a problem when you go to try to actually stain with your antibody of interest. The other type of formaldehyde-based fixative is paraformaldehyde, and this is a polymer formaldehyde and to use it at 4% solution, and it has a stain fixing capacity as a 10% formalin solution. In this case, you do not have methanol to breakdown products, so this is considered to be a methanol-free fixation method. So these formaldehyde-based fixatives are actually very, very useful and they preserve tissue morphology very well. The main disadvantage is that the cross-linking of proteins can sometimes prevent the antibody from recognizing the epitope of your protein of interest; and I will discuss later how we can get around this problem.

​So after fixation the tissues need to be further processed by embedding and sectioning them. In terms of embedding, the structural detail by infiltration with a matrix. So this is typically paraffin; a paraffin wax is used. The tissues are dehydrated in sequential baths of ethanol and xylene, and they're finally infiltrated with a molten paraffin wax. Once this wax has hardened, the tissues are cut into thin sections for optimal resolution and transmission of light. The sections are typically about 5 microns thick.

​Another type of tissue processing, instead of going through a cross-linking fixation and then embedding, is freezing the tissue. Freezing can preserve the tissue without any chemical modification. The one disadvantage to this is that ice crystals can form upon freezing, and this can damage the morphology of the tissue. So this is usually addressed by transferring the tissues to a 20% sucrose solution, and the sucrose here acts as a cryoprotectant. So following that step, the tissue can now be snap frozen and this occurs in a bath of isopentane on dry ice. This will provide an optimal temperature for quickly freezing the tissue to minimize damage. At this point, the tissue is then embedded in a gel, usually OCT and cut in a cryotome. This is then followed by a fixation with an organic solvent such as acetone. Methanol and ethanol can also be used, and these function to fix the tissue by dehydration precipitation.

​At this point, the frozen tissues are ready to undergo the IHC protocol. But going back to our paraffin-embedded sections, these will still require further processing. The slides must be de-paraffinized and rehydrated, but because the paraffin wax can interfere with the actual staining of the tissue with the antibody, it must be removed completely. This is accomplished by applying serial incubations of clean xylene. This is followed by multiple washes in ethanol, and gradually bringing the tissue to an aqueous solution of distilled water. The final step in preparing these paraffin-embedded sections is antigen retrieval. So, as I mentioned before with the cross-linking fixatives, formalin or PFA, the cross-links can sometimes mask the epitope. So to reverse these cross-links we use a technique called antigen retrieval; this is also known as epitope retrieval or de-masking. The two most common methods of antigen retrieval are a heat-induced retrieval as well as an enzymatic retrieval.

​In heat-induced retrieval the sections on the slide are heated in a buffer, and there are two main buffers: the first being a sodium citrate buffer at a low pH of six, or a Tris-EDTA buffer, and this is a higher pH of nine. The sections in the solutions are then heated, and this is accomplished either in a pressure cooker, some people use a vegetable steamer. Additionally, a microwave or autoclave have also been used. ​The next type of retrieval is enzymatic retrieval, and in this case the tissues are actually incubated with an enzyme such as trypsin, pepsin or proteinase K, and this will also function to reverse cross-links and make the epitope more accessible.

​So now that we understand how the tissue is processed, and how it's prepared for IHC, let's go through the actual IHC procedure. The first step is a blocking step, and this is important because it will prevent any non-specific binding and allow for the antibody to specifically recognize the protein of interest. Typically, a serum-based block is used, a 10% serum and that serum, usually we recommend, comes from the animal that the secondary antibody was raised. This will minimize background and allow for a more specific antibody antigen signal to be observed. This step is usually performed for 1 hr at room temperature.

So following blocking it's now time to incubate your sections with the primary antibody, and this will target your protein of interest. Very often antibodies are provided with a dilution range that is optimal, however, it is definitely up to the end user to determine what the optimal concentration for the antibody is, especially if they're using a different tissue than what the antibody has been tested on. So, it's really important that optimization is taking place here. We recommend using a range of dilutions, also outside of the recommended dilution range. As you can see here on the left, we have very good contrast and you can distinctly see the brown stain and the counterstain. Here on the right though there's clearly too much antibody being used. This is a lot of background staining and cannot be distinguished from the specific stain, so this is a very important step.

​Following the incubation of the primary antibody, which we usually recommend go overnight at 4°C, comes some washing steps. You want to wash off all of the unbound primary antibody before incubating with the secondary antibody. You may also need some additional blocking steps at this stage. This will depend on the conjugation of your secondary antibody. The secondary antibodies are typically conjugated to an enzyme or a fluorophore. With regards to enzymes, HRP or horseradish peroxidase, or alkaline phosphatase known as AP, are used. It's important, because the tissue that you are staining may contain endogenous peroxidase or phosphatase activity, so it's important to block that at this stage. This can be accomplished with a peroxide block for HRP or using a chemical such as levamisole as an alkaline phosphatase block. If your secondary is conjugated to a fluorophore, this additional blocking step is not necessary. In addition, some antibodies are biotinylated and this serves to amplify the signal, which I will discuss in greater detail later on. But if you're using a biotinylated antibody, you're also going to need an endogenous biotin block.

​So once that blocking step has been performed, you can now incubate the sections with your secondary antibody. This is required only if your primary antibody is not already conjugated. The conjugation is important for the actual detection of your signal. We usually recommend this step to go for just about an hour at room temperature. The dilutions can vary widely, and additionally at this stage a different dilution should be tested.

​Following incubation with the secondary antibody, again you want to wash off any unbound antibody. Once you've performed those washes, if you've used an enzyme-based detection system, you're going to need to add your substrate chromogen. With regard to HRP, this is usually DAB33 diaminobenzidine and a brown precipitate will be the positive, and this remains the antibody antigen site through electrostatic interaction. This step is not required if you're using a fluorescently-tagged secondary antibody. Those can be observed immediately under the microscope, and you need to use a special fluorescent microscope that will have filters to excite the fluorophore and allow for transmission of the signal.

​So in order to highlight the different detection methods, I'd like to point your attention to this cartoon image of detection. So here we're looking at in both cases the chromogen and the fluorophore that you have your antibody, or rather your protein of interest. Your primary antibody that will target that protein. You would label secondary antibody that will recognize the primary antibody. So if you're using a mouse primary antibody you'd want to use an anti-mouse labelled secondary antibody. On the top I'm looking at the chromogen, and you have advanced to your enzyme HRP. You then add your substrate DAB, and this is converted into a brown-colored product. This is a very stable reaction and it can be stored.

​On the other hand, with the fluorophore, the secondary antibody is going to be conjugated to that fluorophore, and it's going to need special light to excite and emit a signal. So you must be aware of the different filter capacities of your fluorescent microscope, and choose a fluorophore for which you have a filter so that you can observe this signal. In the case of fluorescence, this is not a stable signal and the fluorescence signal will eventually photobleach from the light and dissipate, so this is not as permanent and cannot be stored long-term.

​Finally, the last step is to just coverslip and then you can do further observations under the microscope. The purpose of the coverslip is simply to protect your section, and it also improves optical quality when you're looking at it under the microscope and taking images.

​Now that we have gone through the IHC procedure, I'd now like to discuss some variations on that procedure. The first being amplification, so in terms of amplification this is particularly useful when your antibody has a low affinity for the protein of interest, or if the protein is expressed at a very low level. So in order to amplify that signal, here I'd like to direct your attention to the section and we're looking at a rat brain stained with tyrosine hydroxylase. This is followed with an anti-rabbit IgG conjugated to biotin, and I mentioned biotin earlier in the talk, and this was then followed by an avidin-HRP. So the way this works is that biotin and avidin have a very strong affinity for one another, and you have your biotinylated secondary antibody and they're usually 3-6 biotin modules per antibody. This can in turn be bound by a conjugated avidin or a complex of avidin and biotinylated reagent. So here we see your secondary antibody that's biotinylated. You also have your avidin marked here with the black X that is down to the biotin, which is also conjugated to HRP. So this is going to bring a lot more of your enzymes to the site of the protein, and this will result in a higher degree of precipitate and the colored, so there'll be more color at this site. So this is really great to allow visualization of a signal that you may not have gotten with just a HRP or a fluorescent method of detection.

​The second variation that I'd like to discuss is double immunostaining. So here we're looking at a double staining in fluorescence, so we're looking at a mouse olfactory bulb transfected with GFP, and an anti-GFP antibody was used to detect that; so here you see that in green. This is combined with an anti-Tbr2 antibody, so this is shown here in the pink or purple, and the blue is a DAPI stain, to stain the nuclei. So for double immunostaining, fluorescence is usually preferred because the signals are very robust and can be distinguished from one another very easily. You can utilize antibodies from the same or different species; with the same species you want to use a conjugated primary antibody and this will allow for direct detection. If you are using antibodies - primary antibodies - they'll have been raised in the same animal, you want to use a sequential staining method. So your first stain with your GFP antibodies, for example, and then you can go in with your red stain.

​The final variation that I want to discuss is non-protein antigens. Antibodies can be generated against antigens other than proteins, and these include carbohydrates, fatty acids and inorganic molecules. So here we're looking at a section that had been tissue-treated with a BrdU, and this is incorporated into newly-replicated DNA. We went in with a BrdU antibody, so you see that in brown here and, of course, this is a counterstain of hematoxylin. So these antibodies have been used for detection of stains other than proteins, and this can be very useful.

​So now that I've gone through the basic IHC protocol and some variations of that, I'd now like to discuss some troubleshooting tips. There are three common problems that researchers typically face: the first is that there's no signal at all; the second is a high background signal; and the third is just an incorrect signal altogether. So let's begin with no signal, so you've gone through the long process of performing your experiment, collecting your tissue, preparing it and then undergoing the IHC procedure. When you look at it under the microscope you don't see anything, and this obviously can be very frustrating. So this indicates a few possibilities: the first is that perhaps you need to further optimize your protocol; the second being maybe you're using a defective antibody, or finally that your protein of interest may not actually be expressed in the sample that you're testing. So there's a number of different ways that we could distinguish among these possibilities, and the first being antigen retrieval. So often researchers choose one method of antigen retrieval, and I find that works with most or all antibodies. This is likely, but not necessarily guaranteed, so each antibody may require a separate method of antigen retrieval. Some may prefer using a low pH buffer, while other antigens prefer a high pH buffer. So I definitely recommend that you try multiple methods of retrieval, unless one has specifically been stated on the datasheet of the product.

​The next way to increase your signal is to increase the antibody concentration, so, again, antibodies are usually provided with a dilution range. However, it's always a good idea that this be optimized by the end user, and they try multiple antibody concentrations. The duration of the incubation period is also important here; we do recommend going overnight at 4°C. Some labs routinely do this step for only 10 to 15 minutes, or just a couple of hour at room temperature. So increasing that antibody concentration as well as the length of the incubation time, will definitely matter.

​Finally, you may want to consider an amplification step. If you're not sure that your protein is expressed at a high level, this is a great way to help enhance that signal if possible. Of course, you always want to include a positive control, a tissue where you know the protein is expressed and, hopefully, robustly enough that it's easily detected.

​So the opposite problem is also encountered frequently by researchers, and that's a high background. In this background, essentially it overwhelms the specific staining and so your specific stain cannot be distinguished from all the background. The first thing we always recommend is to reduce the antibody concentration, this will often help to reduce that background. The other thing to mention is that perhaps going for overnight at 4°C may be too long of an incubation period, and certainly seen antibodies that in just 15 minutes produce a really nice signal, so it's also recommended that you try multiple incubation times.

​The next thing you want to make sure that you're doing is performing the necessary blocking steps. So, as I mentioned, we typically block using a normal serum and this is serum from an animal that has not been immunized. It's important that actually comes from the animal that the secondary antibody was raised, and this will greatly help to reduce any non-specific binding. You also want to make sure you're sufficiently blocking for any endogenous enzymatic activity, so this will include peroxidase, alkaline phosphatase and biotin. You want to make sure that you are performing those steps, and that those blocks are for long enough that they will actually function to block the endogenous activity. We also recommend optimizing the antigen retrieval protocol. So, as I mentioned, even in a case of no signal not all antigen retrieval methods are compatible with all proteins and antibodies, and some actually don't require any antigen retrieval at all. But it's always a good idea to try different types of retrieval to see which one will be best for your staining.

​Finally, we always recommend an isotype control to isolate the problem. You want to be sure that there's nothing innate about the antibody or the isotype of the antibody that's resulting in signal. In addition to that, we also recommend performing a no primary control and this will help determine if the problem is at all related to your secondary antibody. If you are getting a background signal from your secondary antibody, then we typically advise that you use a pre-adsorbed secondary, so this will prevent any non-specific binding to your tissue.

​The third problem that you might encounter is just an incorrect signal altogether, so you're looking at your tissue under the microscope and you were expecting to see a cytoplasmic stain, and instead you see a nuclear stain. Unfortunately, in the case of incorrect signal, this usually cannot be resolved by protocol modifications, and it's very rare that that may be the case. So one thing you want to always do is verify, and the easiest way to do that is running a western blot. As you can see here on the left you're looking at the bad blot, and this is for a Chd7 and you can see multiple bands on the block that you don't really see anything at the molecular weight; predictive molecular weight of the protein. So in this case, since the antibody clearly does not seem very specific to the target. However, here on the right you're looking at a very clean western for beta actin. You see just a single band at the correct molecular weight, so this is a much better antibody.

​To conclude this portion of the talk, I would just like to say that immunohistochemistry is constantly evolving and we're continuing to develop new methods of fixing tissue, antigen retrieval and signal amplification strategies. For additional information, protocols and troubleshooting tips I'd like to refer you to the following websites. Of course, there's the Abcam website and we have protocols for immunostaining, as well as most other antibody-based techniques. This is a very useful resource, and we include the protocols as well as common problems, and troubleshooting tips. You also should feel free to call us at any time if you have any difficulties with one of our products, or if you have any questions about a protocol.

​The next resource is IHCWorld, again, this is a great resource: protocols and troubleshooting tips, and a forum to discuss issues you may have with a product. There's some other websites that are very good is Histonet and the Proteinatlas, which, of course, has images of staining for most proteins and various tissue types; and Gensat, which is useful for neural protein stains.

​I would also like to direct you to a couple of different books: the Immunohistochemistry - Methods Express Series, edited actually by our own Simon Renshaw in the lab over in our UK office. Additionally, there is Histotechnology and Histological, and Histochemical Methods, and these are all really great resources if you have questions, or would like more specific information about how to stain your tissue.

​Before I take any questions, I just wanted to introduce you all to two new and exciting product lines offered by Abcam: the first are Expose IHC kits. The Expose kits are very useful for a number of reasons: first of all, it's a biotin-free IHC detection method. This will eliminate any long polymer backbones or bulky molecules, and instead the enzyme is conjugated to the antibody by long-arm linkers. Because the molecule is smaller you get much greater sensitivity, because it can more easily diffuse into the tissue and reach the primary antibody and this, of course, will increase the amount of target detected. There is an improved signal to noise ratio, as compared to other detection methods and there are flexible IHC options, so these kits are very adaptable to the current detection methods that you may already be using. We have HRP/DAB, HRP/AEC and AP red detection systems, so this is especially useful and you can look at different colors or if there are multiple stainings in a single tissue. Finally, the important thing to know here is that they're compatible with both mouse and rabbit primary antibodies. I should mention that they are also meant for paraffin-embedded sections.

​So let me first go into how does the kit work? I'm going to direct you here to the right at the rabbit primary antibody image, and as you can see you have your protein of interest, the antigen, a rabbit primary antibody; so this is the antibody you wish to use to detect your protein of interest. The kit then provides a goat anti-rabbit HRP, so this will recognize the rabbit primary antibody. As you can see here, there are multiple HRP molecules with these long-arm linkers, and you simply add your DAB substrate and this results in the brown precipitate. As you can see here, there is an opportunity for multiple reactions to take place enhancing the signal. This is easily adapted to a mouse primary antibody.

​So, again, you have your protein of interest on the left, your mouse primary antibody and then the kit includes a rabbit anti-mouse. So this is key to the adaptability of the system to a mouse primary antibody. So you have a rabbit anti-mouse which will be recognized by a goat anti-rabbit, which is HRP conjugated. Again, you see these long linker arms bringing multiple HRP modules to site, and by simply adding your DAB and producing that brown precipitate. As you can see, it's a very easy system to use and very effective, and I would encourage you to check out our website and we do have customer reviews for some of these kits, and some of the data is very beautiful. So I would definitely recommend you take a look at that, and potentially this is something that you want to try in the future.

​The other product line that I'd like to introduce are our EasyLink conjugation kits. These are especially useful if you're looking for a primary antibody and you can't find the one that's conjugated to the fluorophore enzyme that you want, and you can easily do this yourself. There are several advantages to our EasyLink kits: number one, use of the kit will eliminate indirect protection methods. As I mentioned, you can directly label your primary antibody so you don't a secondary antibody anymore. This kit also eliminates any column separation steps, so very often with conjugation you have to further purify after you've labeled the antibody. Our system works in only a single tube, single step and there is no risk for loss of material, because you're not transferring your antibody into multiple different tubes and then going through columns for purification. So you still maintain the amount of the antibody that you put in. This also eliminates any non-specific binding of secondary reagents, so this is especially useful if you're performing mouse-on-mouse staining, for example, and you want to avoid the detection of any mouse IgG. You can simply conjugate your primary antibody as you no longer have to worry about that problem. Finally, it does eliminate additional incubation steps and sample dilution, so you cut back on time and getting your results much more quickly.

​Importantly, these kits also have uses outside of immunohistochemistry. The labelling kits will also facilitate the study of multi-protein complexes, advanced multiplex immunoassay technologies, the use of antibodies generated from either the same or a different species, so this can very useful when you're performing double immunostaining; and if both of your antibodies are raised in a mouse, if they are conjugated to different enzymes and fluorophores, they can be used together. Finally, the identification of optimal antibody pairs in ELISA, so, as you can see, there are multiple uses for these kits.

​How does the kit work? As I mentioned, the main reaction occurs in a single tube, so first you have your primary antibody, you then add a modifier and this solution is then transferred into the conjugate. Once you have that setup, it's actually just a 3 hour incubation at room temperature and your antibody is labeled within that time. 3 hours later you add your quencher to stop the reaction, and within 30 minutes you have a labeled antibody that is ready to use. I should note that once labeled, these antibodies can be stored for later use at 4°C.

​So there are a couple of other elements I would just like to draw your attention to that are important for using these kits. The first is that the conjugation process is based on a number of important factors: the first being buffer compatibility. Often, antibodies are supplied in buffers that may contain different reagents that are not compatible with the kit. So, for example, if you have a high degree of sodium azide or a lot of BSA, this can actually inhibit the labeling process. Fortunately, we also have antibody clean-up kits available that can help eliminate or reduce the amount of the unwanted reagent.

​Additionally, the amount of antibody is important, you do require a certain amount of antibody and we do have different sizes available to meet any of your needs. If, for some reason, the antibody that you have is not very concentrated, we also have antibody concentration kits and this will allow for the sample to be used in the conjugation kit for labeling. Finally, of course, the binding of the antibody is also going to come into play here. So the label is added in excess, and that's going to depend greatly on what the label is, so it's in a ratio of 1:1 or 1:4. Again, this ratio is going to be based on the type of label that's being used and it's different for enzyme conjugation versus fluorophore, molecular weight and the amount of antibody. I would like to point out that the label is attached to the antibody by a covalent bond, so this is perfectly stable and you don't have to worry about non-dissociation of the label and the antibody.

​Finally, I'd just like to point your attention to this link. When you download the protocol, or rather when you download this presentation at the end of my talk, you can easily find this link on our website and it will give not only the advantages that I described here and how the kit works, but also some frequently asked questions. So, hopefully, your question can be answered there and, if not, we encourage you to give our technical support department a call and will be happy to assist you with that.

​The last thing I wanted to note is just that the kits have numerous conjugates available, so we have enzymatic detection, so you have alkaline phosphatase, glucose oxidase and horseradish peroxidase. We also have avidin and biotin and streptavidin, as well as a whole range of fluorescent molecules. So we can pretty much accommodate whatever you're looking to label your primary antibody with.

​So with that, I would like to conclude this presentation and I'm happy to take any questions that you may have. I have a question here from Kate Simmons: What is a pre-adsorbed antibody? A pre-adsorbed antibody is one that has been incubated with tissues from multiple different samples and species, so that anything that might react to that species or that tissue is eliminated. This will allow for a more specific signal when you use the antibody in your detection.

​So another question from Mike: How long should I fix my tissue? Generally with fixation the rule is 1 hr per mm of tissue - this is generally acceptable. You may also want to talk to colleagues or check the literature for fixation recommendations for your tissue. The 1 hr per mm of tissue is a good guideline to follow, but it doesn't hurt to see what other people who are working with the same tissue type are doing.

​Another question I have here, let's see, from Ambrin: Does increasing an incubation period with a primary or secondary increase the signal intensity? Yes, this is definitely going to be an important factor. As I mentioned, we typically recommend incubating overnight at 4°C and this is plenty of time to get a nice, clean signal. However, I've also seen a beautiful signal after 10 to 15 min of incubation, but if you're having difficulty getting a signal or the intensity of the signal, increasing that incubation period just allows more time for the antibodies to find and bind to your protein of interest.

​I have a question here from Nicole: Can your conjugated antibody be stored for later use? Yes, definitely, and once labeled we do recommend storage at 4°C, but it doesn't all have to be used right away and every time you need a conjugated antibody you don't have to remake it every time. Different labels will have different shelf lives, so we just recommend checking the product datasheet for more specific information about the kit you're interested in.

​Let's see what else we have. So from Anthony: Can you use antibodies that are already conjugated to a fluorochrome so that you can perform a single-step staining, instead of a two-step antibody stain? Yes, absolutely. This is essentially the simultaneous method of double immunofluorescence, so both antibodies, both priority antibodies can be incubated with the tissue together. This only really presents a problem if you are using a primary antibody that's not conjugated, or two primary antibodies that are not conjugated and they're raised in the same species. So with that, and once you put your secondary into the mix it's going to bind to both antibodies. You won't be able to distinguish the signal, so in that case you'd want to use a sequential method of staining, and we have some really nice protocols for both sequential and simultaneous double immunostaining on our website.

​A question from John: What is normal serum? Normal serum is serum from an animal that has not been immunized, so we use this for blocking because we wouldn't expect any of the antibodies that you're using in your staining to be present in that serum.

​Another question from Elena: How do I select the right secondary antibody for my experiment? Well, when it comes to selecting a secondary antibody there's two things you want to keep in mind: the first being, what species your primary antibody raised in. So if your primary was raised in a rabbit, you want a secondary that is anti-rabbit. The other thing to keep in mind is the isotype of your secondary antibody. So antibodies can come in various different isotypes, in general, there is the IgG1, IgG2, 2a and 2b, IgM, so you just want to make sure that you're aware of what the isotype is. Because if you're using an IgG2a primary antibody, you want to make sure you're not using an IgM secondary antibody, because they're not going to cross-react.

​I'm just looking here for another good question. Another question from Ambrin: How can we check the specificity of an antibody? It's really difficult to actually do this with IHC, because unless the localization is incorrect, it's difficult to know if the antibody is specific or not. So we recommend the easiest way to do that is to just run a quick western blot. As I showed earlier in the presentation, this will allow you to easily see if multiple bands are being picked up, and some multiple proteins potentially are being recognized by the antibody. So if you're seeing a lot of non-specific bands, perhaps your antibody is not that specific. There's always a risk of some cross-reactivity with a polyclonal antibody, so you may see fainter bands, but, in general, you should see a nice, single clean band and that should help confirm your results in your IHC experiments.

​Do you guys have a trial Expose kit? This is from Nicole. Well, as far as trial kits go we actually - instead of offering trial sizes with all of our products, we place a guarantee and this is known as our AbPromise. So all products are guaranteed to work as they have been specified on the datasheet, and for any reason you have difficulties with one of our products, simply email us or call us and let us know what the problem is. We'll do our best to help you troubleshoot and see if there are ways we can improve your results, but if that's not the case, as long as you're contacting us within 6 months of purchase, we're happy to replace or refund any product that isn't working. So it's nice that you not only get the kit, but you get 100% scientific support and we're here and happy to help you.

​Let's see, I think we have time for maybe one more question. What fluorescence microscope was used in the examples you demonstrated in this webinar? This comes from Anthony. Actually, all the images of tissue sections that you saw in this presentation were submitted by our customers. So for each of our products we have a system known as AbReviews, and from the datasheet you can submit an AbReview for one of our products. We then review that information to make sure just everything makes sense, and then we do publish that on our website. So we don't change anything in the review, we always ask our customers to clarify anything that may not be clear, but we will publish both positive and negative feedback, and this is a great resource to see how other customers have experienced using our products. So, unfortunately, I don't have any information about the microscopes that were used to collect those images. However, you may - if you ever see something on our website, often customers are fine with being contacted by other customers, and you can certainly direct questions to the customers as well as to our scientific support team.

​I think I got through all of the questions here. In any case, thank you all for submitting your questions and for joining us today. If you have any questions about what was presented, please feel free to contact us and you can give us a call. Our email address is [email protected] and we welcome your questions at any time, and we're happy to assist you. So I hope that you found this webinar to be very useful.

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