Posted in | Immunology | Lab Techniques

IHC/ICC Staining Techniques Using Single and Multiple Labels Webinar

Watch this on-demand webinar presented by our resident IHC/ICC specialist

Review our tips and techniques on how to get the best out of your experiments and overcome the most common technical challenges.

About the Presenter

Simon Renshaw, Abcam Senior Imaging Scientist.

Webinar Topics

  • Sequential staining using antibodies
  • Simultaneous staining using antibodies
  • Multiple stainings using antibodies and cytomarkers
  • Analysis of images and validation

Webinar Transcript

Hello and welcome to Abcam's webinar on IHC/ICC staining techniques using single and multiple labels. Today's principle speaker is Simon Renshaw, Senior Imaging Scientist at Abcam. Simon completed his Biomedical Sciences Degree at the University of Bradford. After this he completed his BMS1 training in the Department of Histopathology at Addenbrooke's Hospital in Cambridge, UK. This was where he was introduced to using immunohistochemistry and immunofluorescence techniques for diagnostic purposes. Simon joined Abcam in 2001.

Joining Simon today will be Judith, our Product Manager at Abcam. Judith completed her Molecular Biology Degree and PhD at the University of Dundee. Upon completing this, Judith moved to the MRC Laboratory of Molecular Biology in Cambridge and she joined Abcam in 2011.

Just to let you know that when you log off from this webinar, you will be redirected to a webpage where a downloadable PDF copy of the presentation can be found. I will now hand over to Simon who will start this webinar.

SR: Thank you, Lucy. Hi everyone, welcome to this Abcam webinar entitled: IHC and ICC staining techniques using single and multiple labels. As the title suggests, I'm going to talk you through all of the important factors and steps that must be considered when performing both single and multiple label immunostaining. Later on in the presentation we'll be doing a three-question quiz just for fun, so watch out for that one folks!

Before I begin, it is worth noting that a lot of the important aspects of designing IHC and ICC experiments were covered in the webinar: Optimizing IHC and ICC results through careful experimental design. The video of this can be found in the Abcam blog. Today's webinar focuses more on the practical aspects of performing immunostaining, both with single and multiple labels. However, for completeness certain experimental design stats will be reiterated where appropriate.

We're going to work through and discuss the following in detail. Firstly, the IHC/ICC using a single reporter label both day one and day two staining protocol. Day one will consist of antigen retrieval both heat-induced and enzymatic, buffers and detergents, blocking buffers and, finally, primary antibody incubation. Day two will cover quenching endogenous peroxidases, briefly touch on detection systems, discuss whether to use an enzymatical fluorescent reporter label, and the various chromogens that are available for enzymatic detection. We will then go on to talk about tinctoral counterstains before finally discussing mounting. In case you're wondering, I will talk about fluorescent counterstains in the multiple staining section. In the second part of the webinar, we will look at IHC and ICC using multiple reporter labels by beginning with how fluorescence works, before moving on to combinations of fluorescent reporter labels. We will then talk about fluorescence compared to enzyme reporter labels, followed by a simultaneous staining, then sequential staining, finishing with some discussion about controls.

Firstly, we will go through a generic IHC or ICC protocol suitable for use with a labelled secondary antibody, polymer, or avidin-biotin-complex. As you will have learned in the past webinar, there are numerous variations in each step, depending on your experimental design. I will talk you through each protocol step, and, when needed, I will discuss any common experimental design variations. The focus for the last part of the webinar will be the design of experiments using multiple labels.

We'll go into these in much more detail, but it's worth just quickly talking through the individual steps and the protocol, just to give you an idea of what's to come. First of all, on day one, any pre-treatments are performed such as de-waxing of paraffin sections and antigen retrieval. There is then a wash stage followed by a blocking non-specific protein/protein interactions, or, in other words, non-specific staining, after which there's another wash. The primary antibody is then incubated on the specimen overnight at 4 °C.

We then go on to day two, and the first job is to wash off the primary antibody before blocking any endogenous peroxidases; if, indeed, you're using a peroxidase enzyme reporter label. After this it's time to start introducing the detection system and allowing it to incubate before washing it off. If the detection system you're using is ABC-based, you would add the ABC complex at this point. Let it incubate and wash it off, but, if not, you'd skip this step. Similarly, if you're using an enzyme reporter label, you'd then incubate in chromogen before washing it off, but, if not, you'd also skip this step. You'd also block any endogenous phosphatase at this point, if you're using a phosphatase enzyme reporter label. Any counterstains would then be applied before washing off the excess. If you're using an enzyme reporter label and chromogen combination that is not alcohol soluble, you would next incorporate and dehydrate, clear and mount procedure. But, once again, if not, you'd also skip this step. Finally, whatever happens, you're going to want to mount your stained specimen in some way in order to help preserve it before, during and after microscopic analysis.

Let's now discuss the first step of the protocol in a little more detail, which is concerned with performing any required specimen pre-treatments such as de-waxing of tissue sections, and antigen retrieval. Antigen or epitope retrieval serves to break the methylene bridges formed during aldehyde fixation, allowing antibodies access to certain fixation-sensitive antigens. Antigen retrieval is, therefore, only necessary on specimens that have undergone aldehyde fixation, and only then if a fixation-sensitive antigen is being demonstrated. All specimens should be on coated slides to help prevent disassociation. Both frozen and paraffin sections usually survive antigen retrieval if mounted on coated slides, unless they're particularly friable, such as adipose tissue. There are two common methods of antigen retrieval: heat-induced, or HIER for short, which involves the use of a suitable buffer, such as trisodium citrate pH6 or EDTA pH9. The other is enzymatic, which involves the use of a suitable enzyme solution such as trypsin, pepsin, pronase, or proteinase K. Both methods can be incorporated into automated systems either as part of the immunostaining platform, or a standalone antigen retrieval vessel. But if done manually, heat-induced antigen retrieval is commonly performed in a pressure cooker or microwave, and enzymatic in a water bath.

The following antigen retrieval protocols are generic, concentrating on the above methods using trisodium citrate of pH6, the heat-induced antigen retrieval and chymotrypsin for enzymatic. However, please remember that there is no universal antigen retrieval solution. For each antigen the most appropriate antigen retrieval solution, or enzyme, pH, method and duration must be established.

So for performing heat-induced antigen retrieval, you begin by pre-preparing the trisodium citrate buffer by mixing together 5.88 g of trisodium citrate, 44 mL of 0.2 M hydrochloric acid and 1,956 mL of ultrapure water. This would then be adjusted to pH 6 with 1 M of sodium hydroxide and hydrochloric acid, before being poured into the pressure cooker using enough volume to cover the slides by about 1 cm.

The pressure cooker itself would then be put onto a hotplate and the lid placed on top, and by this I mean don't secure it down. The hotplate would then be turned on to bring the buffer to the boil.

While waiting for the pressure cooker to come to the boil, dewax and rehydrate any paraffin sections by placing them in a suitable rack and then three changes of xylene for 3 min each, followed by three changes of alcohol for 3 min each, followed by cold-running domestic supply water. Keep them in the running water until the pressure cooker comes to the boil. With dewaxing the xylene removes the wax, the alcohol then removes the xylene and finally the water removes the alcohol. In other words, the slides are going from an organic to an aqueous phase using alcohol as an intermediate phase, since both organic and aqueous liquids are mixing with the alcohol.

Once the buffer is boiling transfer the slides from the running water to the pressure cooker, taking care with the hot solution and steam. Secure the pressure cooker lid following the manufacturer's instructions, and once the cooker has reached full pressure set a timer for 3 min. When the 3 min has elapsed turn off the hotplate and place the pressure cooker in an empty sink.

Activate the pressure release valve and run cold domestic supply water over the cooker to aid depressurization. Once depressurized open the lid and run cold domestic supply water into the cooker for 10 min, again, taking care with the hot solution and steam. You are now ready to continue with the immunostaining protocol.

Please note that this protocol can be modified with regards to buffer used, pH and duration of antigen retrieval. 3 min duration is only a suggested starting point; less than 3 min may leave the antigens under-retrieved, and more than this it may leave them over-retrieved which may cause false or background staining, and may increase section disassociation from the slides. Optimization is therefore advised, retrieving slides to 1, 3, 5, 10 and 20 min before being immunochemically stained. This protocol can easily be modified for use in the microwave, but always begin retrieval timings from when the buffer is boiling. If you choose to use a microwave it's advisable to use a scientific one with a temperature probe and a stirring function, in order to prevent the retrieval solution from boiling vigorously; thus aiding section disassociation and to eliminate cold spots. Finally, and probably most importantly, please follow the manufacturer's instructions for the safe use of a particular pressure cooker or microwave that you have in your lab. Please also follow health and safety guidelines for any of the chemicals that you are using.

Let us now look at enzymatic antigen retrieval protocol. Begin by setting a water bath to 37 °C. Next, obtain two troughs large enough to accommodate the rack slides and add a sufficient amount of ultrapure water to each trough to cover the slides. Place the troughs into the water bath and allow the temperature of the ultrapure water to warm to 37 °C.

De-wax and rehydrate any paraffin sections as outlined in the heat-induced antigen retrieval protocol, and place them into one trough of ultrapure water at 37 °C to warm.

Remove the other trough of ultrapure water and to this dissolve 0.1 g of calcium chloride, and 0.1 g of chymotrypsin for 100 mL of ultrapure water, using a magnetic stirrer to ensure that all reagents are properly dissolved.

Once dissolved, bring the solution to pH 7.8 using 1 M sodium hydroxide and hydrochloric acid before returning the trough to the water bath. Allow this enzyme solution to reheat at 37 °C.

Transfer the warm slides into the enzyme solution for 20 min, and then remove them and place them in cold-running domestic supply water for 3 min. You are now ready to continue with the immunostaining protocol.

Chymotrypsin features in this protocol, but any enzyme can be used, just ensure to adjust the Vmax temperature and pH accordingly, pH 7.8 and 37 °C being those for chymotrypsin. Pre-warming the slides in ultrapure water avoids reducing the temperature of the enzyme solution when they are placed within it. Ensure to make up the enzyme solution fresh and use it straightaway, because any delay could affect its proteolytic properties. Similarly, ensure that the enzyme solution reaches its Vmax temperature before introducing the slides. As with heat-induced antigen retrieval, less than 20 min may leave the antigens under-retrieved, and more than this, may leave them over-retrieved which may cause false or background staining, and may increase section disassociation from the slides. Optimization is, again, therefore advised, retrieving slides to 5, 10, 15, 20 and 25 min before being immunochemically stained. Most importantly, please also follow health and safety guidelines for any of the chemicals that you are using.

We have now completed the first step in our protocol and we are about to move on to the second, which is the first of many washes in buffer containing detergent. A common question regarding buffers is generally what is best to use? Well, again, there isn't one universal buffer; you have to use a one most suitable according to your experimental design parameters. As a rough guide, I would personally recommend TBS containing 0.5% Triton X-100 for tissue sections, and PBS containing 0.1% Tween for cytological preparations. TBS is of higher ionic strength than PBS; TBS, therefore, helps to give cleaner background staining, but is obviously unsuitable for cytological specimens as it can osmotically cause cells to lyse.

Detergents, or more specifically in this case surfactants, such as Tween and Triton improve antibody penetration by removing lipid from cell membranes, therefore, permeabilizing them. A demonstration of most antigens certainly benefits from detergent being present in the buffer, however, please be aware that some cell membrane proteins may be stripped out using a detergent so it is best to emit it in such cases. Detergents reduce surface tension helping reagents spread out over the specimen, and are also considered to dissolve Fc receptors in frozen sections, reducing background staining.

Triton is more aggressive than Tween, making Tween more suitable for cytological specimens since they tend to be more friable. I found that an addition of 0.1% Tween in buffers used for cytological preparations cause a gentler to be of permeabilization suitable for most antigens.

If more rigorous permeabilization is required, 10 minutes incubation in 4 mM sodium deoxycholate can be used between protocol steps one and two, with an additional buffer wash before step two. The concentration of detergent can simply be increased also, but pay attention to potentially negative effect, as previously described. Incidentally, if you're using a phosphatase label avoid making it up in PBS, since the phosphates in the buffer will inhibit it.

After the first wash, we now move on to the blocking step. Blocking buffer helps prevent unwanted background staining that could mask positive staining, or lead to false positive results. This is achieved by incubating in buffer containing detergent, plus 10% normal serum, 1% BSA and 0.3 M glycine for 2 h at room temperature. Use normal serum from the species used to raise a secondary antibody. Any endogenous immunoglobulins in the specimen that have affinity with the secondary antibody, will be pre-adsorbed by the immunoglobulins in the serum. BSA serves a similar purpose by saturating any proteins that will react non-specifically with other proteins such as antibodies. In aldehyde fixed specimens, glycine binds to any free aldehyde groups reducing the incidence of hydrophobic protein-protein interactions.

The image on this slide shows an example of what can happen when the wrong serum is used. Serum from the same species of the secondary it was raised against has been used in opposed to serum from the same species from which the secondary was raised. Hence, a secondary antibody in red binding non-specifically over the entire specimen.

We have now performed blocking, and after another wash step we will move on to addition of the primary antibody. The primary antibody is applied to the specimen, optimally diluted in buffer containing detergent plus 1% BSA. The primary antibody will target the epitope it is raised against. Ensure that the primary antibody is raised in a different species to that of the specimen, in order to reduce background staining from a secondary antibody binding to endogenous immunoglobulin. This isn't really a problem when using cytological specimens, unless you're staining cells with a B-cell lineage, which is unlikely since they are non-adherent by nature.

Once the primary antibody has been added to the specimen, the next step is to incubate it overnight at 4 °C. Lower affinity antibodies are allowed more time to bind in an overnight incubation. Overbinding isn't an issue, since once the antigens are saturated no further binding can occur. The reduced temperature helps to reduce background staining by increasing reaction times, and decreasing the incidence of non-specific protein-protein interactions. The effects of this can be enhanced by gentle specimen agitation from an orbital shaker. An overnight incubation is not usually essential, but has the advantages outlined above. You will have hopefully already performed an optimization experiment with regards to the optimal primary antibody dilution.

After all of that, congratulations! Day one is finished. Time to go home and relax to arrive all fresh and ready to tackle day two. Our primary antibodies have been incubating overnight, and we're now back in the lab to do day two of our immunostaining protocol. This begins by giving our slides another wash to remove the primary antibody, before going on to blocking endogenous peroxidases; if we're using a peroxidase enzyme reporter label that is. If not, you just skip steps two and three, going straight on to step four.

Blocking endogenous peroxidases only applies to detection systems that use a horseradish peroxidase, or HRP, reporter label. Red blood cells contain endogenous peroxidases, which if not quenched with hydrogen peroxide, will react with the chromogen alongside the peroxidase reporter label, producing a false positive staining. Incubating paraffin-embedded specimens in 1.6% hydrogen peroxide, made up in buffer containing surfactant, for 30 min at room temperature, or 5 min for a frozen section of cytological preparation adequately blocks endogenous peroxidase activity without having a detrimental effect on tissue epitopes. That is why endogenous peroxidase quenching is done after primary antibody binding, in order to ensure that this isn't an issue. You should only use fresh hydrogen peroxide, since it quickly breaks down into water and oxygen at room temperature, making it ineffective at quenching. It is good practice to store hydrogen peroxide frozen and thaw directly before use. Aside, when staining tissues high in endogenous peroxidases such as spleen, it is sometimes best to use a non-peroxidase reporter label which is alkaline phosphatase or AP. Quenching of endogenous phosphatases will be discussed later.

The next step - step four - is to begin incubating the specimen with the chosen detection system. This protocol focuses on three popular systems: those being a directly reporter label conjugated primary antibody, a biotinylated secondary antibody, if an ABC, Avidin Biotin Complex or a secondary antibody reporter label complex in the form of a dextran polymer or compact polymer. I went into how each of these systems work and the subject of signal amplification in my last webinar, including the pros and cons of each, so I'm not going to dwell on this here, but you can check it out on the Abcam blog.

Please note that if using an ABC system, there will be an additional step - step six - which I will talk about in more detail later, where the actual ABC complex is added. So at this stage - step four - it will just be the biotinylated secondary antibody that is added. In general, ensure that all of the detection system reagents are optimally diluted in buffer containing surfactant, plus 1% BSA and incubated on the specimen for 1 h at room temperature.

This is a poignant place to discuss whether to use an enzyme reporter label or a fluorescent one? A very common dilemma is whether to use a fluorescent or enzyme reporter label in a particular experiment. Enzymatic reporter labels are commonly used on tissue sections, and fluorescent reporter labels are commonly used on frozen tissue sections and cytological preparations. However, this is not absolute; the detection system should be tailored to suit the immunostaining experiment. The main benefit of fluorescence over enzymatic is that all of the fluorescent channels can easily be viewed separately, and then merged to form a pseudo-colored image. It is, therefore, easy to see signal colocalization between a fluorescent counter stain and that of the detection system, without any specialized spectral or mixing software. Weak signals from the primary antibody can be also observed in isolation without any interference from other signals.

Often tissue sections display autofluorescence due to some tissue components being naturally fluorescent, such as collagen. Formaldehyde fixation also increases the degree of autofluorescence. If strong enough it can mask the signal from fluorescent reporter labels, making results interpretation difficult. Enzymatic detection is therefore more appropriate for tissue sections. Cytological preparations and frozen sections are commonly enough exposed to formaldehyde for long enough to exacerbate autofluorescence, and cytological preparations often do not possess such naturally fluorescent components.

Our detection system is now incubated for 1 h and we'll wash it off with a buffer rinse. If we're using an ABC system, it's at this stage that it will be incubated on the specimen. Make this up according to the manufacturer's instructions. The ABC system is used to provide as Avidin alone, with a Biotin label complex in a separate container. End users must mix both accordingly, and wait for at least 30 min for complex formation before adding it to the specimen, or it would not function to its full capability as a detection reagent. However, if you are not using an ABC system, you'd skip step six and seven and proceed directly to step eight.

If you are using an enzyme reporter label, you are now ready to add the chromogen. Reporter labels that are enzymatic in nature, produce a stable colored precipitate for the site of primary antibody binding when exposed to a suitable chromogen. Amongst several, the two most popular enzyme labels for immunochemistry are horseradish peroxidase and alkaline phosphatase. Each of these has commonly used chromogens, namely AEC, DAB and DAB containing nickel for HRP; and fast blue, fast red and new fuchsin for AP. As you can see from the table, the different enzyme and chromogen combinations produce a different color precipitate at the site of antibody binding. Also note that some of the precipitators are alcohol-soluble which has an important significance when mounting, which I will come on to later.

Ensure that all chromogens are made up according to the manufacturer's instructions, and incubate them on a specimen according to manufacturer's guidelines. Typically for DAB, 10 min at room temperature generally gives good results. Always observe chromogen expiry dates as storage conditions. HRP and hydrogen peroxide form a complex in the presence of DAB chromogen with HRP catalyzing the breakdown of hydrogen peroxide into water and oxygen. DAB is oxidized during this process and provides electrons to drive the reaction. It is the oxidized DAB that forms the black/brown colored precipitate to the site of the reaction. Hydrogen peroxide, therefore, plays a key role in this reaction and since it quickly breaks down into water and oxygen at room temperature, DAB kits that have passed their expiry date may be ineffective. Appropriate COSHH guidelines should be observed regarding the storage, use, handling and disposal of any laboratory agents. If using an alkaline phosphatase reporter label add 0.24 mg/mL Levamisole to the working chromogen solution. Levamisole quenched endogenous phosphatase activity, reducing unwanted background staining, although not in placenta or small intestine. Don't worry, the phosphatase reporter label itself will not be affected by the Levamisole.

After washing off the chromogen and running domestic supply water, we can now apply a suitable counterstain. Counterstains add color-contrasted the cells or tissues by staining certain cellular structures, thus helping to define the localization of the immunostaining. They can be tinctoral or fluorescent in nature, complementive in section system used to visualize a primary antibody. For enzyme detection systems, tinctoral and nuclear counterstains are commonly used. I'll talk more about fluorescent counterstains in the second section of this webinar.

The most common nuclear counterstaining used when employing an enzyme chromogen detection system, is Hemotoxylin. Hemotoxylins are available in numerous formulations identified by the type of mordant used, and whether they stain progressively or regressively. Hemotoxylin stains cell nuclei various shades of purple or blue, according to the type used. Hemotoxylin, or specifically it’s oxidation products, hematin is an ionic and, therefore, has little affinity for DNA. Mordants are iron salts which combine with hematin, creating a positively charged dye-mordant complex with the ability to bind an ionic chromotin. Alum or aluminium mordant hemotoxylins can be used progressively or regressively.

Progressive hemotoxylins, for example, are Mayer's, Gill's or Carazzi's can be applied to tissues or cells until the desired degree of nuclear staining is observed. Progressive hemotoxylins such as Harris's, are applied to tissues until over-staining is observed before having a proportion of the excess staining removed by immersion in acid, such as 1% acid alcohol. This process is called differentiation. This makes progressive hemotoxylins simpler to use and regressive due to the emission of the differentiation step, and the subsequence compatibility with alcohol-soluble enzymes, substrate end products, such as that produced by the reaction of HRP and AEC.

All hemotoxylins, both progressive and regressive, are blue to a desired level of staining has been achieved. Hemotoxylin stained nuclei red under acidic conditions, however, in an alkaline environment hemotoxylin turns a pleasing blue/purple color. Running domestic water, supply water is commonly used for this purpose. It usually has sufficient alkalinity to achieve this, especially in hard water areas. In soft water areas, an alkaline solution can be used for bluing such as 0.05% ammonia.

Other commonly used tinctoral nuclear counterstains are light green, fast red, toluidine blue and methylene blue; then in nuclei the green, red or blue, respectively. One important consideration when using a nuclear tinctoral counterstain, is not to make the staining too intense if you are demonstrating a nuclear antigen, since the counterstain can potentially mask a positive signal from the detection system.

Let's now discuss specimen preparation for microscopic analysis. It is essential to prepare the immunostain specimen in order to preserve it while being imaged during long-term storage, and to enhance image quality. This typically involves placing a glass coverslip over the specimen, securing it in place with a suitable adhesive known as mounting media. Mounting media can be either aqueous, suitable for both fluorescent and enzymatic labels, or organic suitable only for enzymatic. Organic mounting medias tend to set hard, allowing the glass coverslip to remain securely in place.

Refractive indexes are better with organic mounting media, such as DPX giving a much sharper, crisper image down the microscope. However, ensure that the color precipitate forms from the reaction of the enzymatic label with a substrate is compatible with organic mounting media. For example, the reaction of peroxidase and AEC is alcohol soluble, so will disappear during the dehydrating and cleaning process, if an organic mounting media is used.

Fluorescent labels require aqueous mounting media; 10% glycerol can be used, but commercially available medias containing anti-fade reagents are superior. When using a fluorescent label, it is advisable to microscopically observe the mounting media on a blank coverslip to ensure that it does not produce any autofluorescence. I'm happy to say that our immunostaining specimen is now ready to be analyzed down the microscope. Microscopic analysis is a complete webinar on its own, especially to go into how to setup a microscope optimum illumination compared with light microscopy and fluorescence; and so I won't be going into that right now.

Let's now think about multiple staining. The purpose of immunostaining using multiple reporter labels, is to simultaneously visualize the cellular localization of two or more antigens in the same cell or tissue section. A different reporter label is used for each antigen in order to distinguish them apart. Antigens may be co-localized, meaning within the same cellular compartment of any given cell, or separate.

When two or more antigens are co-localized, the color of the separate reporter labels will mix to produce a new color. Both fluorescent and enzyme reporter labels can be used, but these need to be carefully selected to ensure that they do not interfere with each other, and that they are easily interdistinguishable. Similarly, the detection system reagents must not cross-react or interfere with each other in any way. This webinar will concentrate on fluorescence multi-staining, that way there's always the potential for another separate webinar for enzymatic later.

Before we go on, we will first consider how fluorescence works. A fluorescent molecule has the ability to absorb light of a specific wavelength and to re-emit light at a longer wavelength. It loses energy in order to achieve this by interacting with its environment prior to the emission of fluorescence; a phenomenon called internal conversion, which is the loss of energy in the absence of light emission.

Electrons can exist either at a ground state or resting state called S0, or in excited states of higher energy called S1 and S2. At each of these electronic states the fluorochromes can exist in a number of vibrational levels called 0, 1 and 2. There is not enough energy at room temperature to populate the excited electronic states of S1 and S2, or the higher vibrational levels of the ground state. Therefore, absorption generally occurs from molecules in the lowest vibrational energy state, and only in the presence of light.

Following absorption, a fluorochrome is usually excited to a higher vibrational level and be the S1 or S2 before relaxing to the lowest vibrational level of S1, thus completing the process of internal conversion. This combined with the emission of a photon results in fluorescence. A fluorochrome can repeat the excitation emission cycle many times before excitation bleaches the fluorescent signal, otherwise known as photobleaching. However, some fluorophores photobleach more readily than others. FITC, for example, can repeat the excitation emission process approximately 30,000 times before photobleaching occurs. Second generation fluorochromes, however, such as the Alexa Fluor® range do not photobleach as rapidly, and are generally brighter since they have a higher extinction coefficient making them more appropriate for multi-labelling experiments, as demonstrated in the images on this slide.

With this in mind, we will now discuss how to go about choosing combinations of fluorescent reporter labels for a multiple staining experiment. Unless the appropriate spectrum or mixing software is available, fluorochromes must be selected so that their emission spectra do not overlap. This is critical, otherwise the emission of one fluorophore may be detected in a filter set intended for another fluorophore. This phenomenon is known as bleed through or cross-talk, or crossover. Bleed through occurs due to the non-symmetrical spectral properties of many fluorophores, and associated wide bandwidths.

Consider simultaneous spectral emission analysis of Alexa Fluor® 488 and Alexa Fluor® 555 in the lower right image. The maximum emission peaks are seemingly well-defined and separated, but the maximum emission peak for Alexa Fluor® 555 there is still significant overlap with that of Alexa Fluor® 488. This creates bleed through of Alexa Fluor® 488 into the Alexa Fluor® 555 channel. This same phenomenon can be seen in action in the top right hand image with Cy3® bleeding through into Texas Red®.

The aim, therefore, is to minimize the area of emission overlap by using another fluorophore of long enough emission wavelength to minimize the overlap, or to eliminate it. If Alexa Fluor® 594 were used in place of Alexa Fluor® 555, the bleed through effect of Alexa Fluor® 488 will be small enough for both dyes to be used together in a multiple label experiment, since the emission profile of Alexa Fluor® 594 sits further to the right of Alexa Fluor® 488 than Alexa Fluor® 555, since the area of emission overlap is smaller.

This is why three or four colors fluorescence using one fluorophore from separate areas of the spectrum is so popular. Since the absorption and emission spectra are adequately far enough apart to allow no or very little bleed through. For instance, a traditional dye combination of DAPI for blue, FITC for green, TRITC for yellow and Cy5® for red is commonly used. An equivalent second generation dye combination of this would be DAPI for blue, Alexa Fluor® 488 for green, Alexa Fluor® 555 for yellow and Alexa Fluor® 647 for red.

It is worth mentioning at this stage that not every fluorescent signal needs to come from a conjugated antibody. It could be from a nuclear counterstain such as DAPI, from an organelle-specific stain such as MitoTracker, or from a cell membrane counterstain such as wheat germ agglutinin conjugated to a fluorescent label. Carefully selected microscope absorption and emission filters can drastically help to reduce bleed through. Indeed, it is a filter set on the use of the microscope that will ultimately dictate the fluorophores that could be successfully used.

There are several steps that can be taken when designing a multi-labelling experiment using fluorescent reporter labels to help minimize or eliminate bleed through. Firstly, use a spectral analyzer program to observe the absorption and emission spectra of your potential fluorophores, alongside the characteristics of your microscope filter set to assess probable compatibility issues. Try and use fluorophores with as narrow emission characteristics as possible, to minimize overlap with other fluorophores of longer wavelength. Stain the least abundant protein for the fluorophore with the highest quantum yield. If two or more proteins are expected to be of similar abundance, try and reduce the concentration of the fluorophore with the shortest wavelength to try and lessen the degree of bleed through into the ones with longer wavelength. Balance the filter sets for the microscope carefully to match the spectral absorption and emission profiles of each fluorophore as closely as possible.

Balance the exposure and neutral density filter settings of the microscope to fine tune overly aggressive fluorescent signals. Finally, before conducting the actual mordant staining experiment, observe each fluorophore separately in situ using the filter sets of the other fluorophores being used to practically assess any bleed over issues. If necessary, adjust experimental design within parameters described above.

Although this webinar isn't focused on multiple immunostaining using enzyme reporter labels, I thought it would be a good idea to briefly compare enzymatic to fluorescence. As we have been discussing, fluorescence has a wide-range of available reporter labels and has been long proved to be extremely sensitive, with each label being observed in isolation with relative ease. However, it has several disadvantages when compared to enzymatic. Firstly, when in close proximity to each other, fluorochrome signals can be quenched due to the transfer of energy from an excited fluorochrome to another, turned resonance energy transfer. Secondly, fading or photobleaching of the fluorescent signal can occur during storage or analysis. Lastly, specimen autofluorescence is exacerbated by aldehyde fixation, potentially interfering with results interpretation.

Enzyme and chromogen combinations allow for two or more end product precipitate colors to be used negating the problems associated with fluorescence. Also, since the colored precipitate stays localized on the tissue independently of the detection system, this allows to the elution of primary and detection system antibodies by heat-induced epitope retrieval, allowing subsequent rounds of immunostaining to occur without any fears of unwanted antibody cross-reactivity. With both fluorescence and enzyme chromogen products, the evolution of spectral unmixing software allows the isolation of signals even when co-localization is indistinguishable with the human eye, allowing numerous different labels to be used.

Let's now discuss actual multiple immunostaining techniques. Simply put, a multiple staining experiment is a combination of two or more individual single label staining techniques. The protocol discussed in the first half of this webinar is a good basis for a multi-staining protocol, in search of the necessary incubation on wash steps where appropriate. The use of highly cross-adsorbed or pre-adsorbed secondary antibodies is highly recommended in all cases, to help minimize any cross-reactivity. Optimizing the specific concentrations, etc., of each of the single labelling techniques in isolation, is strongly recommended before combining them in a multiple labelling experiment. The main concern is how to avoid the individual components of each of the separate detection systems from cross-reacting.

Multiple staining techniques themselves can be broken down into two groups: simultaneous, where the primary antibodies are applied together, followed by the section systems; and sequential, where one single label staining technique is completed before the other.

We shall begin by taking a look at simultaneous staining. If the species used to raise the primary antibodies are phylogenetically different enough, they can be applied to the specimen together. A primary antibody combination raised in mouse and rabbit would be considered sufficiently phylogenetically different, whereas a combination raised in mouse and rat would not. Secondary antibodies raised in the same species against those species bearing different fluorophores, can then be applied at the same time. Simultaneous staining can also be achieved using primary antibodies that are raised in species that are not sufficiently phylogenetically different, even raising the same species if the antibodies are directly labelled with a different fluorescent label per primary antibody. Of course, this is only viable if all of the antigens are very abundant and a low degree of signal amplification is required. Simultaneous staining can be performed using primary antibodies that are raised in the same species, if they are of different isotypes and isotype-specific secondaries are used. As you can see, it is quite limited in what you can do in simultaneous staining, especially in terms of signal amplification, which is where sequential staining is more advantageous.

Sequential staining is much more flexible than simultaneous staining, allowing additional signal amplification to be performed rather than just secondary antibodies, for use on low-abundance antigens. The most sensitive detection systems should be reserved for the antigen that's expected to be the least abundant. Particular attention needs to be paid to the order that the reagents are added. An example would be three primary antibodies all raised in mouse: one directly conjugated to a fluorophore, one directly conjugated to biotin and one on conjugated.

Step one: incubate the specimen with the unconjugated mouse primary.

Step two: will be to incubate the specimen with a fluorophore-labelled secondary, such as goat anti-mouse.

Next, incubate the specimen with mouse serum to ensure that the goat anti-mouse antibodies are saturated.

Step four: is to incubate the specimen with the biotinylated mouse primary.

In step five: we would then incubate the specimen with a fluorophore-labelled ABC complex.

Finally, step six: is to incubate the specimen with the directly fluorophore conjugated mouse antibody.

This would result in three color staining with no potential for the reagents to cross-react. Please also note that it will be buffer washes in between each step in the sequence above. Another method when using antibodies raised in the same species, is to technically change the species of one of the primary antibodies by adding a bridging antibody of some description. An example would be two primary antibodies, both raised in mouse: one on conjugated and one directly conjugated to HRP.

Step one: incubate the specimen with the first unconjugated mouse primary.

Step two: will be to incubate the specimen with a fluorophore-labelled secondary, such as goat anti-mouse.

Next, incubate the specimen with mouse serum to ensure that the goat anti-mouse antibodies are saturated.

Step four: is then to incubate the specimen with a second HRP conjugated mouse primary.

In step five: we would then incubate the specimen with an anti-HRP antibody, such as a rabbit anti-HRP.

Finally, step six: is to incubate the specimen with a fluorophore conjugated goat anti-rabbit antibody.

Alternatively, the second mouse antibody that has been directly biotinylated and an ABC system used. Again, please also note that there will be buffer washes in between each step in the sequence above.

Lastly, let us consider the controls that must be employed when doing multiple immunostaining. As with all immunostaining techniques, controls are essential to validate that any staining is a true result, and not from an intrinsic property of the detection system or specimen. Since multiple staining is a combination of two or more individual single label staining techniques, the number of individual reagent controls that could be used for each detection system can be rather daunting, especially given the time constraints of the modern laboratory. At a minimum, the following should be done. Firstly, positive controls performing the separate single staining techniques of each primary antibody, and detection system combination. Lastly, negative reagent controls performing separate single staining techniques for each detection system, minus the primary antibodies.

My part of this webinar is almost over, but with regards to multiple immunostaining, if you remember nothing else remember the following. Numerous staining combinations are possible as long as careful attention is paid to the experimental design. Select fluorophores so that their emission spectra do not overlap and match them to the filters on your microscope. Carefully consider the various detection systems available, the potential cross-reactivities and the order that the reagents are added. Finally, always use the appropriate controls and perform each separate staining technique in isolation, in order to optimize a concentration of each reagent and to assess potential bleed through.

Thank you very much for your time and I hope that you found it useful. I'll be back in a few minutes to answer some of your questions, but in the meantime over to you Judith.

J: Thank you Simon for such a detailed seminar, and hello again. I would like to take this opportunity to tell you a bit more about Abcam's IHC/ICC resources and products that will help you to improve your cell imaging experiments. Rabbit monoclonals, or RabMAbs, offer high affinity and specificity, which results in high sensitivity and low background staining. This makes them ideal reagents for use in demanding applications such as IHC on formalin-fixed or paraffin-embedded tissues. Best experience which RabMAb has been tested on multiple human tissue arrays for IHC, or multiple samples for ICC/IF. RabMAbs also offer a diverse epitope recognition of human protein targets and the mouse orthologs, so there is no need to generate a separate surrogate antibody. Due to the fact that they are rabbit-generated, they're ideal for use in mouse or red tissue samples. It can also be easily paired with mouse or rat monoclonal antibodies for simultaneous staining. For further information, please visit

For staining of your RabMAb we recommend one of our recently released Alexa Fluor® conjugated secondary antibodies. These antibodies are available conjugated to Alexa Fluor® 488, 555, 594 and 647. All of these secondary antibodies have been extensively tested in the Abcam laboratories to guarantee bright staining and low background. The selection of pre-adsorbed antibodies is large to ensure those species cross-reactivity. The dilution range for all products is between 1/200 and to 1/1000, giving you roughly 250 stainings. In addition to that, all products are competitively priced. For more information, please go to

Abcam's catalogue includes a whole range of cell imaging tools for multicolor staining. Discover our CytoPainter range of kits for staining of actin filaments, mitochondria and lysosomes in multiple colors. It is an easy way to study co-localization without having to fiddle around with multiple antibodies. CytoPainter kits can be used in combination with secondary antibodies and nuclear dyes. An example of this is the IHC image of mouse embryoid bodies shown on the top right corner.

For trouble-free staining of nuclei, why not try far red dyes that will display nuclear staining in just five minutes. Included in this range are DRAQ5 and DRAQ7, which can be used for staining live or fixed cells. The bottom image of nuclei is stained with DRAQ7, the blue staining originates from AMCA conjugated secondary antibodies.

We have an extensive portfolio of validated secondary antibodies for cell imaging, including our pre-adsorbed Alexa and dialyzed conjugated secondary antibodies for minimal cross-reactivity. Our range also includes Chromeo conjugated secondary antibodies for step microscopy, and AbGold conjugated secondary antibodies for high resolution electron microscopy. In order to increase tissue penetration in your IHC experiments, we recommend you try one of our F(ab’)2 antibodies.

For non-fluorescent imaging and enzymatic detection, Abcam also offers a comprehensive range of products for IHC. Included in the portfolio are EXPOSE IHC kits, which provide greater sensitivity in comparison to polymer and ABC detection systems. This is achieved through a smaller detection complex. Our IHC portfolio also includes the classical biotin and streptavidin kits, in addition to various reagents. If you would like to know more about our cell imaging products, please visit On this page you can find more detailed information, and can zoom in onto some of the images.

A complication often experienced when using mouse antibodies and mouse tissue, is the high level of background. This is due to the secondary antibody binding endogenous mouse IgG. A product we offer that provides a solution to this is the Mouse on Mouse IHC detection kit. This polymer-based detection kit contains a blocking reagent to block endogenous mouse IgGs, ensuring minimal background in addition to a simple and reliable protocol. More about this product can be found at

As Simon mentioned during the webinar, it is important to prevent photobleaching. For that purpose, Abcam offers a range of fluoroshield mounting reagents. These are available with or without nuclear dyes, such as DAPI and propidium iodide. Abcam scientific support team is here to answer any questions you may have. The team members are multilingual and offer support in a range of languages, including French, Spanish, German, Chinese and Japanese. You can contact them in the US, UK, Hong Kong and Japan.

On the Abcam website you can find a variety of free resources and protocols. Of potential interest to you are the new IHC applications, and the 'Understanding secondary antibody' guide. The latter explains when to use F (ab’)2 fragments and pre-adsorbed secondary antibodies in multiple staining experiments.

Also, I would like to highlight our future webinars. We would be delighted if you could join us on March 21st for a webinar on Advanced Immunoprecipitation. This webinar will be presented by Dr Sean Garry and Dr Rachel Imoberdorf from Abcam. It offers lots of hands-on advice and troubleshooting tips to improve your IP experiments. For our Spanish-speaking audience, we offer on April 10th a Western Blotting introduction webinar. You can also listen to all of our past webinars, including Simon's recent webinar on designing IHC/ICC experiments, by simply going to

To thank you for attending this webinar, we would like to give you a special 25% discount on all secondary antibodies, CytoPainter kits, IHC kits, avidin and streptavidin, and RabMAbs. All you have to do to take advantage of this offer is to quote promotion code ICCTBDYY when placing your order. I would like to finish by thanking Simon and all of you for attending, and would like to wish you all the best for your future IHC-ICC experiments.

SR: Thank you, Judith. As with the first webinar, we've had some really excellent questions coming through. Due to time constraints I can only go through about three of them, so I've picked out three really good ones. Kevin has asked: I have high background in my multi-labelled experiments. Why could that be? Well, it could be due to so many reasons, Kevin, such as over-retrieving antigens, maybe try less retrievable time, a different buffer, more enzyme. Insufficient blocking, so too short a blocking time, or the wrong serum's used. Your tissues could be rich in endogenous enzymes, or they've not been sufficiently quenched. Tissue could also be rich in endogenous biotin if using an ABC detection system. What else? Species cross-reactivity, so the detection reagents could be cross-reacting or the secondaries could simply be binding to immunoglobulins in the blocking serum. Maybe you want to try using pre-adsorbed secondaries for additional specificity. The antibodies that you're using may be unsuitable for IHC or IF, or may also generally just be non-specific, and there could also be high levels of autofluorescence in your specimen. I don't know your exact experimental design.

There are numerous other possibilities too far, really, to cover right now, but if I were you I'd go into all your protocol line-by-line and see if you can identify any of the above possibilities. Don't forget to include all the necessary controls for each of the single-staining procedures being used, and perform each of them in their entirety in isolation. That may well hold the key to where your background staining is originating. Anyway, good luck with that!

Another question. Mandy says: I am using donkey serum for blocking and anti-sheep secondary, my background is really high, what can you suggest? Well, the most obvious thing that springs to mind is that even though your secondary antibody is raised against sheep immunoglobulin, it's also detecting the donkey to some degree. It's all to do with this, with phylogeny, so donkey and sheep are both ungulates - correct me if I'm wrong - and for that reason, the immunoassay sequence and the immunoglobulin heavy chains will be quite similar, hence, a possibility for cross-reactivity.

I guess there's several things that you can do. Number one, you can use an anti-sheep which has been pre-adsorbed against donkey, and see if that makes a difference. Or you cannot use donkey serum at all for blocking, omit it completely and see if the problem stops. Or, I guess, as well you can see if you can find a primary antibody towards your antigen, which is not raised in sheep; so if you use a different secondary. Try and find a mouse or rabbit primary, for instance, and use a secondary that is raised in goat, for example. Give those a go and see if they help.

We've just got time for one more and the last one is from Steven, and he'd like to know: You mentioned spectral or mixing software, could you tell us a bit more about this? I'm going to be completely honest, Steven, I haven't had much experience of actually using it myself, but I've attended several lectures where people have been very impressed with it, and have shown some of the good results that they've obtained with it in action. Believe me, if I controlled the department's budget, I'd buy one tomorrow. So, in a nutshell, it's microscopy software that is much more sensitive to color than the human eye. In other words, it can distinguish between very subtle color differences in labels, especially when they're merged in co-localization experiments, allowing much easier separation and visualization of the signals. This, therefore, let's you use reporter labels that spectrally overlap both chromogenic and end products, and fluorescent labels, allowing for additional labels to be used in a multi-labelling experiment that otherwise you just wouldn't have been able to distinguish apart with the human eye. It's well worth the money, if you've got the money to spare. As I said, due to time constraints they're the only questions I can go into, but brilliant questions. Thank you very much. Over to you Lucy to wrap things up.

Thanks Simon and Judith. As Simon mentioned, unfortunately we've ran out of time and we have not been able to answer all the questions received, but for those whose questions that have not been answered, our scientific support team will be in contact with you shortly with a response. Also, we've received some questions about where people can find a copy of the presentation slides. When you log-off from the webinar you will be redirected to a webpage where a PDF can be downloaded, and also you can find out more information about the webinar promotion. If you do have any questions about what we have discussed in this webinar or have any technical enquiries, please don't hesitate to contact our scientific support team who will be very happy to help you. They can be contacted at [email protected] Finally, we hope that you have enjoyed this webinar and found it useful for your work, and we hope to welcome you to another webinar in the future. Thank you again for attending, and good luck with your research.

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