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We take you step by step through the major pitfalls of designing and conducting these types of assays.
Simon Renshaw, Abcam Senior Imaging Scientist.
Hi everyone, welcome to this Abcam webinar entitled: Optimising IHC and ICC results through careful experimental design. As it says, I'm going to talk you through all of the important factors that you must consider when planning your IHC or IF experiment, in order to get the best possible outcome. So the factors that you must consider can be roughly broken down into the different specimen formats that are available: fixation methods, processing tissue into paraffin wax, antigen retrieval, detection systems, controls, counterstains, specimen preparation for analysis and, finally, we'll pull everything together with a quick caption summary. Firstly, I thought it would be good to establish exactly what an antigen is, since it's mentioned in numerous places throughout this webinar. An antigen, which is short for antibody generator, is an entity that is detected as foreign: meaning, not normally present in the host biological constitution by the host's immune system.
The host immune system will then produce antibodies against the antigen, labelling it for destruction. Essentially, anything could be an antigen, such as viruses, bacteria, pollen, certain chemicals, etcetera. However, it is a specific area on any given antigen that is recognised by an antibody, termed the epitope. In the case of proteinaceous antigens, the epitope consists of a specific short sequence of amino acids on the protein surface. So for the purpose of this webinar, that is what is being referred to by the term 'antigen'. I thought it would also be good to clarify exactly what the difference is between IF, ICC and IHC. IHC, or immunohistochemistry, is immunological demonstration of an antigen on a tissue section. ICC, or immunocytochemistry, is immunological demonstration of an antigen on a cytological preparation. Both use a reporter label system in order to visualise the binding of the primary antibody. The reporter label can be either an enzyme, which produces a coloured precipitate in the presence of a suitable substrate, or fluorescent.
Enzyme reporter labels are commonly used for IHC, and fluorescent for ICC. However, this is just common practice, and in most cases both types of reporter label can be used for either application. IF, or immunofluorescence, therefore simply refers to immunological assay that uses a fluorescent reporter label. Although, for the reasons outlined above, it is commonly assumed that ICC is the implied application when referring to IF. The very first step to optimising an immunostaining experiment, is to properly understand the antigen that you're attempting to demonstrate. Investigating what tissue has the antibody already been used to detect the antigen, what other tissues are expected to express the antigen? Is the antigen expressed in normal tissues or only in certain disease states? Is expression of the antigen gender or species-specific, or only present in a certain subset of individuals?
Similarly, look at which somatic laboratory cell lines the antibody has already been used on to detect the antigen. What other somatic laboratory cell lines are expected to express the antigen? Does the cell line need exposing to certain conditions grown on a certain support matrix, such as collagen, or need treating with a certain chemical in order to express the antigen? Does cell confluency affect antigenic expression, for example, adhesion proteins exhibiting increased expression where cells touch? In what specimen formats has the antibody already been successfully used with to demonstrate the antigen, such as paraffin embedded, frozen, cytological, etcetera? In which species has the antibody already been used to successfully detect the antigen, and in which species is it expected to cross-react? In which cellular compartment is the antigen expected to be expressed; is it organelle-specific? What fixative or fixatives have been used to successfully demonstrate the antigen? What was the concentration formulation of the fixative solution? How was fixation formed in relation to the specimen and for what duration, and at what temperature?
Has antigen retrieval already been proved necessary to demonstrate the antigen in IHC or IF? If so, was it heat-mediated or enzymatic? Was it performed in a water bath, microwave, pressure cooker or other vessel? Which buffer solution or enzyme was used, and at what pH and formulation? At what temperature was antigen retrieval performed, and for what duration? At what concentrations or dilutions has the antibody already been used? Finally, what species is the antibody itself raised in? All of these above questions will need to be answered in order to optimise IHC or IF staining. This is all well and good, but how exactly do you go about finding this information? Well, if possible, always buy an antibody from a well-established commercial company that is renowned for offering well-characterised antibodies, and good after-sales technical support; and, yes, indeed, Abcam would be a good example.
Generally speaking, the more characterisation information and antibody datasheet it has, the more faith the end user can have that it is indeed specific for the antigen it was raised against. However, this is not always possible and any unknown information can be researched, commonly by using the internet. For example, if your antibody is monoclonal, a search using the clone number can often reveal scientific papers and images demonstrating successful use of the antibody. Such sources will often provide information regarding tissue used, antigen retrieval and working conditions and dilutions. Performing general searches for the antigen in question can reveal sources such as bioinformatics databases that harbour useful information regarding tissue expression, and subcellular localisation. A few examples of these are present on this slide. If the amino acid sequence of the immunogen is provided on the datasheet, a blast search can be performed to give percentage similarity of the antigen in various species to help assess species reactivity. Calling the technical services departments of the commercial company from whom the antibody was purchased, can often prove very useful.
We will now move on to consider different specimen formats. Paraffin embedded tissue sections are tissues that have been fixed, processed and then embedded in paraffin wax to provide support whilst sectioning; otherwise known as 'microtomy'. Paraffin sections provide optimal balance in preservation of morphology and antigenicity. They are very stable when needed to work with, but require de-waxing before immunostaining. They may also require antigen retrieval if fixing the crosslinking aldehyde fixative. They are typically used at 4 micrometre thickness; thicker sections may have the effect of trapping antibody, leading to false/positive staining. Frozen tissue sections are fresh tissues that have been snap-frozen, typically in liquid nitrogen called 'isopentane' to reduce ice crystal artefact, and then directly embedded in OCT, which stands for optimal cutting tissue or temperature, as some say.
Like with paraffin wax, the OCT provides support to the tissues whilst sectioning. They are ideal for antigens that do not withstand standard aldehyde fixation, and paraffin processing. Antigens are in a more native state due to this, but morphology is often poorer, however, the effect of this is minimal if they are optimally prepared. Frozen sections are technically more demanding to prepare than paraffin embedded sections, and are more fragile making antigen retrieval more risky if an aldehyde fixative is being used. They are typically used at 4 to 10 micrometre thickness, like with paraffin sections, thicker sections may have the effect of trapping antibody leading to false/positive staining. Free-floating tissue sections are tissue sections that either begin as frozen or vibratome sections, and are floated out on a suitable buffer where immunostaining directly occurs. This allows for thicker sections to be used, typically at around 40 micrometres, since there is no surface between the section and its support medium to trap the antibodies.
They are more awkward to work with than the frozen or paraffin sections, since they're not mounted on a glass microscope slide during immunostaining. They are very popular with neuroscientists. Cytological preparations can either be whole individual cells commonly in the form of conventional smears, or cytospin preparations, or Thinprep monolayers on microscope slides. Somatic cell lines can also be grown on chamber slides, glass coverslips or 96-well imaging plates. Generally, they have the same pros and cons as frozen sections, but antigen expression in somatic cells lines is often far removed from that of the in vivo subjects, due to a high degree of mutation. For example, HeLa cells, when compared to those from an actual endocervical smear. So let us now consider fixation. It is imperative that tissues, or more specifically antigens to be demonstrated by immunochemistry, are preserved and stabilised in as much as an in vivo state as possible. This is achieved by fixation.
The object of fixation is to preserve cells and tissue constituents in as close a life-like state as possible, and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition, and stabilises the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Fixation is commonly achieved by immersing the excised tissue in a suitable fixative solution that penetrates the tissue, and biologically inactivates proteins by creating conformational changes in the tertiary structure. Some antigens last only minutes after the tissue is excised, or the donor has expired, due to the cessation of biological processes and the homeostatic controls that are governed by them. Once the tissues are removed from the body, they will go through a process of self-destruction. This process is known as autolysis, which starts soon after cell death creating an enzymatic attack, causing the breakdown of protein and the eventual liquefaction of the cell. Subsequent putrefaction, which involves additional breakdown of tissue components by bacteria, further antigen degradation and general loss of tissue morphology and architecture.
Autolysis is more severe in tissues which are rich in enzymes, such as the liver, brain and kidney. The take-home message regarding fixation is that it should provide for the preservation of tissue substances and proteins. It is, therefore, the first step, the foundation step in a sequence of events that accumulates in the final microscopic examination of a tissue section. You simply cannot immunochemically demonstrate an antigen that isn't there to begin with. The first common group of fixatives are the aldehydes, sometimes referred to as non-coagulative or crosslinking, such as formaldehyde, paraformaldehyde and formalin. Aldehyde's fixed by forming covalent methylene bridge crosslinks between lysine residues on the external surfaces of adjacent proteins. The cytoplasm is transformed into a proteinaceous gel-like network, and the cell is respectively rendered in stasis in as much of an in vivo state as possible.
Soluble proteins become covalently bonded to insoluble proteins. Cellular components, such as lipids, carbohydrates and nucleic acids are not directly cross-linked by formaldehyde, but are instead trapped inside a network of crosslinks. It is important to always use neutral buffered formaldehyde to avoid formalin pigment formation, formed from the acidic reaction of formaldehyde with haematin in red blood cells, which is of a dark brown or black colour and can therefore mimic HRP/DAB staining. Glutaraldehyde is commonly only used in tissues destined for electron microscopy, since although the high degree of crosslinking gives a significantly better morphology, the degree of antigen masking is generally far too great for general purposes. The second group of common fixatives are protein denaturing reagents, sometimes referred to as precipitants or coagulants such as acetic acid, methanol, ethanol and denatured alcohol. With protein denaturing reagents, the protein's tertiary conformation is altered by the disruption of internal hydrophobic bonds. The secondary structure is preserved since hydrogen bonds are unaffected. No covalent crosslinks are formed, unlike with aldehyde fixatives, so antigen retrieval is not necessary in protein denaturing reagent fixed specimens. Making them ideal fixatives for antigens that are sensitive to aldehyde's fixation.
Antigens with carbohydrate moieties, such as membrane-bound surface antigens, are commonly fixed with alcohol since carbohydrates are precipitated by alcohol. However, alcohol fixation does tend to hinder CD marker staining. Frozen sections are usually fixed for 10 to 30 minutes in 70 to 95 per cent volume/volume alcohols, in order to help reduce the morphological distortion of nuclear detail and cytoplasmic shrinkage seen with absolute alcohol; and acetic acid and methanol, or ethanol mixture is used for the same reason. Acetic acid also aids alcoholic penetration of the tissue. Alcohol and acetone penetrate tissue poorly, and generally are only used on tissue sections or cytological preparations, rather than pieces of tissue. The take-home message with fixatives is that there is no such thing as a universal fixative, you must use the most appropriate fixative to optimally preserve the antigen in question.
On that note, there are other less common fixatives that are simply a variation on the aldehyde and protein denaturing fixatives that we've just been discussing, such as periodate lysine formaldehyde, Bouin's, acetone, B5, Zenker and zinc formalin. These fixatives have their place in immunostaining, but are only used under special circumstances. I'm not going to go into these in detail, but please research the table on this slide for further information. An example of how critical fixative choice can be is demonstrated on this slide. Here we have some IF staining in ICC on HeLa cells using one of Abcam's alpha-tubulin antibodies, ab7921, detected using ab150105, a donkey anti-mouse Alexa Fluor® 488 conjugated secondary antibody. Antigen expression is expected to be cytoskeletal. In formaldehyde fixed cells the staining pattern isn't all that well-defined, but for methanol it clearly is.
We will now discuss the factors that affect the degree of fixation, and why this is such an important consideration. It is an essential requirement to obtain a delicate balance between under-fixation and over-fixation, which in practical terms tries to balance between good tissue morphology and antigen preservation. There are three parameters that significantly govern this: firstly, it sticks to penetration and this needs to be rapid in order to quickly preserve antigens. Use as small a specimen size as possible, and ensure complete immersion. The current recommendations of a tissue block dimension is to be no greater than 1 x 1 x 0.4 centimetres. Secondly, fixed at concentration, the more concentrated the greater the degree of fixation in any given length of time. Finally, fixation duration and temperature, the longer the duration and the temperature and the higher the temperature, the greater the degree of fixation in any given length of time.
Tissue blocks of the dimensions stated above should also be fixed for a minimum of three and a half hours, and for no longer than 24 hours. The degree of methylene bridge formation increases over time. Under-fixation, therefore, results in poor antigen preservation and morphology. Over-fixation results in a high degree of morphology, but also a higher degree of antigen masking due to the high number of methylene bridge crosslinks. These methylene bridge crosslinks can form a physical barrier to antibody binding, leading to the necessity for antigen retrieval prior to immunochemical staining. Since all chemical reactions are influenced by temperature, the higher the temperature the quicker the degree of methylene bridge formation. For cytological preparations and tissue blocks, 18 to 24 hours formaldehyde fixation at room temperature is regarded as a good compromise between adequate morphological and antigenic preservation.
For frozen sections that have originated from unfixed tissue, 10 minutes formaldehyde fixation at room temperature is suitable. Since the tissue is only around 4 micrometres thick, penetration is not really an issue. Ten minutes provides an adequate degree of crosslinking without over-fixation, since frozen sections are less likely to survive antigen retrieval than paraffin embedded, and cytological preparations even less so. Diluted working concentrations of formaldehyde are typically 10 per cent volume/volume for tissues, and between 4 to 10 per cent volume/volume for cytological preparations. Ethanol and methanol are commonly used at 10 to 95 per cent volume/volume on both frozen sections and cytological preparations, to help reduce the loss of cytoplasm in detail and nuclear distortion often observed with 100 per cent volume/volume alcohol.
Protein denaturing fixatives and acetone are commonly used to incubate frozen sections, and cytological preparations for five to ten minutes. Frozen sections can be fixed at room temperature. Cytological preparations should be fixed with chilled protein denaturing agents or acetone at minus 20°C, in order to help prevent cell dissociation. Frozen sections that have been fixed with 100 per cent volume/volume acetone often have a tendency to be brittle, and display morphological changes such as a loss of nuclear membranes. Such changes can be reduced by leaving frozen sections to dry thoroughly overnight before acetone fixation, and then for a third at ten minutes afterwards before immunohistochemical staining is performed. The take-home message which is quick [unclear 21:09] fixation regime and adhere to it to help maximise antigenicity, and aid reproducibility of results.
Let's now take a look at tissue processing. The next two slides are only applicable to paraffin embedded tissues, but it's a topic that's well worth considering with regards to antigenicity. Blocks of tissue that has been optimally fixed in formaldehyde and are destined to be paraffin embedded, are required to undergo processing which is essentially an aqueous to organic transition to allow them to be impregnated with paraffin wax. It is important to recognise that tissue processing can greatly influence antigenicity. Tissue processing takes around 12 hours in total, around eight hours of this is spent with the tissues incubated in alcohol, around two hours in xylene and then a further minimum of two hours in molten wax. Alcohol itself is a protein denaturing fixative, so will further increase the antigenicity of some antigens. Sorry, my apologies, will further influence the antigenicity of some antigens. It will also completely remove any lipids from the tissue. The latter stages of processing occur at around 6°C, which can also degrade some antigens.
In summary, some antigens may not survive tissue processing very well, or even not at all. In such cases, a frozen section is far more appropriate since it does not have to undergo the same degree of tissue processing. The take-home message here is that it is critical to establish a strict processing regime and adhere to it to help maximise antigenicity. Let us now consider antigen retrieval or epitope masking, as it is often referred. Antigen retrieval; first, reverse the antigen masking effects of aldehyde fixation by breaking the methylene bridges between adjacent proteins, and often physically prevent antibodies from accessing the epitope. As stated, this only applies to specimens that have been fixed in aldehyde fixatives. Some antigens were therefore stained very weakly, or even not at all if antigen retrieval is not performed prior to immunostaining. There are two common methods of antigen retrieval: one is heat-induced, otherwise referred to as HIER, by immersing the tissue sections in a suitable buffer such as trisodium citrate pH6, or EDTA pH9 and applying heat by using a pressure cooker, water bath, microwave or other automated platform.
The other is enzymatic, by immersing the tissue sections in a suitable enzyme solution such as trypsin, pepsin, pronase or proteinase K by using a water bath or other automated platform. There are several factors that affect the degree of antigen retrieval: one is duration; the longer the duration the greater the degree of antigen retrieval. A difference of two to three minutes of antigen retrieval time can have a significant effect on the intensity of subsequent immunochemical staining, and prolonged antigen retrieval times can increase background staining. Temperature is another with heat-induced, if possible, it is best to keep the retrieval solution just below boiling, since boiling encourages sections to dissociate from the glass slide. Using coated slides helps, for instance, APES or poly-l-lysine, since scientific microwaves and automated platforms tend to have temperature control mechanisms to help prevent this. Also, the higher the temperature the greater the degree of antigen retrieval in any given time period. With enzymatic antigen retrieval, the proteolytic action occurred at an optimal level at a certain temperature, so deviating from this may also result in a lesser degree of antigen retrieval in any given time period.
Composition and pH of the retrieval solution also has an effect. Certain antigens appear to prefer one antigen retrieval solution to another, purely down to the composition and pH. With enzymatic antigen retrieval, the proteolytic action occurred is not the small level at a certain pH. So deviating from this may also result in a lesser degree of antigen retrieval in any given time period. The method utilised makes a difference, some antigens appear to prefer enzymatic antigen retrieval and some prefer heat-induced. With heat-induced, the degree of antigen retrieval will vary, depending on the method used. Microwave ovens, for example, come with different power ratings so the degree of antigen retrieval will vary between models on full power over any given time period. Domestic microwave ovens can generate hot and cold spots with the antigen retrieval vessel, leading to different degrees of antigen retrieval according to where the tissue is placed. Again, consistency is a key to achieving reproducible results.
The degree of methylene bridge formation also plays a part. The greater the degree of aldehyde crosslinking within the specimen initially, the lesser the degree of antigen retrieval in any given time period. This, again, demonstrates why a fixation regime is so important. Susceptibility of the antigen to methylene bridge masking needs to be considered. Some antigens appear to require no antigen retrieval at all, and some require a lot. This is down to the amino acid sequence of the epitope and its location within the tertiary structure of the protein. The take-home message is that there's no such thing as universal antigen retrieval solution. Each antigen must be investigated in turn, in order to establish the most appropriate antigen retrieval buffer or enzyme, pH, method and duration. We will now discuss detection systems and reporter labels. I should mention at this stage that if anyone has any questions, then please submit them through the WebEx system for me to answer at the end.
Simply put, detection systems allow the visualisation of the primary antibody found in the antigen being demonstrated. Several common detection system formats are available offering various degrees of signal amplification. Regardless of format, all detection systems involve the use of a reporter label that can be seen by the human eye when the specimen is viewed using an appropriate microscope. Reporter labels can either be enzymatic or fluorescent in nature. Reporter labels that are enzymatic in nature produce a stable-coloured precipitate at the site of primary antibody binding, when exposed to a suitable chromagen. Amongst several, the two most popular enzyme labels for immunochemistry are horseradish peroxidase or HRP for short, and alkaline phosphatase, abbreviated to AP. Each of these has commonly used chromagens: 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 chromagen combinations, there is a different coloured precipitate at the sites of antibody binding.
Also know that some of the precipitators are alcohol-soluble, which has important significance when mounting, which I will come on to later. When selecting the enzymatic reporter label, consider the endogenous enzymes that may be present within the tissue. Endogenous peroxidase is found in red blood cells and can react with the HRP chromagen to produce false/positive staining, which usually can be blocked by applying a solution of hydrogen peroxide to the tissue. Similarly, endogenous alkaline phosphatase activity is commonly found in the large intestine, and can be blocked by incubating the tissue in a levamisole solution. Although, a more practical solution in this case would be to simply use HRP instead of AP. Fluorescent reporter labels, otherwise called fluorochromes or fluorophores, are chemical molecules that absorb light at a certain wavelength, and can re-emit light at a longer wavelength.
Some common examples can be seen here. As you can see, each label has different excitation and emission characteristics. Where possible, I'd advise you to use a second-generation fluorescent reporter label, such as the Alexa Fluor® range since they give a better degree of fluorescence, and they don't suffer from the effects of photobleaching as easily as the likes of FITC, for example. The selection of fluorescent reporter label largely depends on the microscope filter sets, and the choice of fluorescent counterstain. Fluorescent reporter labels and fluorescent counterstains should be selected so their absorption and emission spectra do not overlap, allowing each to be excited and observed separately. Spectral analyser programmes are widely available, but allow the checking of absorption and emission characteristics of the fluorescent labels to ensure that there is no spectral overlap, and to ensure filter set compatibility.
A very common dilemma is whether to use a fluorescent or enzymatic reporter label in a particular experiment. Enzymatic reporter labels are commonly used on tissue sections, and fluorescent reporter labels commonly used are 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-coloured image. It is, therefore, easy to see a signal co-localisation between the fluorescent counterstain and that of the detection system, without any specialised spectral unmixing software. Weak staining 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 a signal from fluorescent reporter labels making results interpretation difficult. Enzymatic detection is therefore often 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 compounds. It is always advantageous to use a signal amplification technique, instead of the directly conjugated primary antibody. This will greatly enhance the visualisation of weakly expressing antigens. Signal amplification typically involves the use of a reporter label conjugated secondary antibody, raised against and, therefore, binding to the species' immunoglobulin subclass of the primary antibody. Multiple secondaries will bind to the primary antibody, greatly enhancing the number of label molecules at the site of binding. Secondary antibodies can be directly conjugated to the label in some way, or be conjugated to biotin. So that's an avidin/biotin complex abbreviated to ABC bearing the conjugated label can be subsequently added.
The following are all very common methods of signal amplification. I'm going to go into each one in a little more detail shortly, so I'm not going to dwell on this slide too much. But, as you can see, each method has one, two or three separate steps and there's a conjugated reporter label in a slightly different way. Out of all of them, dextran polymer, compact polymer and ABC are the most beneficial since these give the greatest degree of signal amplification. Let us now look at the pros and cons of each method in a little more detail. With label conjugated secondary antibodies, each label conjugated antibody will bind to the primary antibody. It is a two-step method primary antibody and secondary antibody, so it's quick to perform. Endogenous biotin isn't a problem, since the detection system does not use biotin. Unfortunately, it offers relatively low levels of signal amplification when compared to a polymer or ABC method. However, amplification is usually good enough to visualise the antigen, especially when using a fluorescent label.
When using an avidin/biotin system, biotin conjugated secondary antibodies bind to the primary antibody. An avidin/biotin label complex then binds in turn to the biotin conjugated secondary antibodies. The degree of signal amplification is often comparable to that of a polymer system, which is a three-step so it's slower to perform than others. Endogenous biotin can also be a problem if not properly blocked. Finally, the ABC system is usually provided as avidin alone with a biotin label complex in a separate container. The end user must mix both and wait for them to form a complex before applying it to the specimen being stained. The ABC complex takes around 30 minutes to form, adding even more time to the immunostaining protocol. It is also worth mentioning that the use of streptavidin gives a cleaner background than avidin, since avidin is positively charged, whereas streptavidin is almost neutral so there is less potential for electrostatic binding.
With the dextran polymer system, dextran labelled polymer conjugated secondary antibodies bind to the primary antibody. This is only a two-step procedure, so it's quick to perform. Endogenous biotin isn't a problem either. Dextran polymer gives significantly higher signal amplification and directly conjugates at secondary, since the dextran backbone allows far more label molecules to be in proximity to the primary antibody. However, the dextran backbone is a relatively large complex and it has been reported that staining of some intracellular antigens is reduced when using a dextran backbone, due to steric hindrance. Finally, we have compact polymer. Sorry, we have compact label polymer systems. Label molecules are covalently bound together in close proximity, then conjugated en masse for secondary antibodies.
Compact labelled polymer conjugated secondary antibodies therefore bind to the primary antibody. Like with the dextran polymer system it is two-step, so it's quick to perform and endogenous [unclear 36:22] isn't a problem. It gives significantly higher signal amplification and directly conjugated secondary, since the compact polymer allows far more label molecules to be in proximity to the primary antibody. The main advantage is that compact polymer is a much smaller complex compared to the dextran labelled polymer complex, greatly decreasing the effects of steric hindrance. This system has very few disadvantages; so few that I couldn't actually think of any, and would therefore be my detection system of choice. Let's move on to look at controls. It is essential that controls are run in an immunostaining assay. Without the appropriate controls, any apparent staining is essentially meaningless. They've searched and verified the staining pattern observed is true, accurate and reliable.
Essentially, there are two types of control: the first are antigen controls; these can be split up into positive and negative antigen controls. With positive antigen controls, specimens that are known to contain the antigen being demonstrated are run alongside the test specimens. A positive result is therefore expected in the positive control. They give assurance that the immunostaining method in its entirety is sound, so positive controls should therefore be treated in exactly the same way as the test specimens. Positive controls are useful when testing tissue specimens of unknown positivity for a particular antigen, when an antibody of no specificity with that antigen is being used. Or when characterised as antibodies of unknown specificity towards a particular antigen, using the same positive control tissue to test all of the antibodies.
Negative antigen controls, on the other hand, are specimens that are known not to contain the antigen being demonstrated: a negative result is therefore expected. Any staining seen in the negative control that it either has to be from non-specific binding of the primary antibody, from some element of detection system, or from an intrinsic property of a test tissue. The second group of controls are known as reagent controls; reagent controls ensure that staining originates from the primary antibody binding to the antigen, and not from aberrant binding of the detection system, or from an intrinsic property of the specimen. False/positive staining from aberrant binding of the detection system, or from an intrinsic property of the specimen can easily be determined by replacing the primary antibody with [unclear 38:53] alone. Any subsequent staining must therefore come from a source other than the primary antibody binding.
Next, we'll look at counterstains. Counterstains have colour contrast cells or tissues by staining certain cellular structures defining the localisation of the immunostaining. They can be tinctoral or fluorescent in nature, matching the detection system used to visualise the primary antibody. For enzyme detection systems, nuclear counterstains are commonly used. With IF, both nuclear and cell membrane counterstains are often used. However, counterstains need to be of a different colour or have sufficiently different absorption and emission characteristics if fluorescent to each other, and the detection systems so the signals can be distinguished from each other. For enzyme detection systems, haematoxylin is commonly used as a nuclear counterstain, either regressively or progressively, producing a pleasing blue/purple colour after bluing. An example of haematoxylin staining can be seen in the top right image on this slide, alongside DAB staining.
Other common nuclear counterstains are light green, fast red, toluidine blue and methylene blue staining nuclei either green, red or blue, respectively. It is important to ensure that the nuclear counterstain isn't too heavy if the antigen being demonstrated is nuclear, or it could mass a positive staining. For fluorescent detection systems there are many fluorescent counterstains available, they can be chemical in nature such as DAPI to stain the nucleus, or the fluorescent molecule such as the Alexa Fluor® conjugated to a lectin, such as wheatgerm agglutinin, used to demonstrate the cell membrane. Both of these can be seen in the image on the bottom right of this slide. Fluorescent counterstains should be chosen according to the filter sets on the microscope, and the absorption and emission spectra of the fluorescent labels being used.
Finally, let's discuss specimen preparation for microscopic analysis. It is essential to prepare the immunostained specimen in order to preserve the specimen and immunostaining while being imaged, and during long-term storage; and to enhance the image quality during microscopy imaging. 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 either be 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 coloured precipitate formed from the reaction of the enzymatic label with a substrate, is compatible with organic mounting media. For example, the reaction of HRP and AEC is alcohol-soluble so it will disappear during the dehydrating and clearing process, if an organic mounting media is used.
Fluorescent labels require aqueous mounting media; 10 per cent volume/volume glycerol can be used, but commercially available media 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 this does not produce any autofluorescence. Let's bring all of this together in the summary. The bottom line take-home message for this webinar are for each antigen the staining processes need to be optimised, and plan your experiment carefully; know what variables you want to try, and how your ideal image should look, what controls you need and always identify any incompatibilities within your chosen immunostaining protocol. Many thanks to all of you for taking the time to listen to my section of this webinar. I will now hand you over to my colleague, Judith, but before I do please remember that I'll be back shortly to answer some of your questions.
JL: Thank you, Simon, for such a detailed seminar. Hello everyone. Just a quick reminder that you can submit questions via the Q&A box on the right hand corner of the screen. 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 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 affinity reagents for demanding applications, such as IHC on formalin 6, or paraffin embedded tissues. To give you the best experience, each RabMAb has been tested on mito [unclear 44:07] areas for IHC, and on multiple samples for ICC/IF. RabMAbs also offer diverse epitope recognition of human protein targets and the mouse orthodox, so there is no need to generate a separate surrogate antibody. Due to the fact that they are rabbit-generated, they are ideal for use on mouse or rat tissue samples. They can also be easily paired with mouse or rat monoclonal antibodies for dual staining. For further information, please visit abcam.com/RabMAbs.
For staining with your RabMAbs, we recommend one of the recently released Alexa Fluor® conjugated secondary antibodies. Currently available are Alexa Fluor® 488 and 647, but from early-2013, Alexa Fluor® 555 and 594 will also be available. All of these secondary antibodies have been extensively tested in the Abcam laboratories to guarantee bright staining and low background. There is a large selection of pre-adsorbed antibodies to ensure low species cross-reactivity. The dilution range of all products is between 1/200, and 1/1,000. Abcam's catalogue includes a whole range of cell imaging tools for [?mito 45:34] colour staining. Discover our CytoPainter range of kits for staining of actin filaments, mitochondria and lysosomes in multiple colours. It is an easy way to study co-localisation 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 the mouse embryoid bodies shown on the top right corner.
For trouble-free staining of nuclei, why not try far red dyes that will do nuclear staining in just five minutes. Included in this range are DRAQ5 and DRAQ7, which can be used for staining of live and fixed cells, respectively. The bottom image of nuclei stains of DRAQ7, the blue staining originates from an [?Abcam 46:26] conjugated secondary antibody. We have an extensive portfolio of validated secondary antibodies for cell imaging, including our pre-adsorbed Alexa and Dylight conjugated secondary antibodies. Our range also includes chromeo conjugated secondary antibodies for spec microscopy, and Ab-gold conjugated secondary antibodies for higher resolution electromicroscopy. In order to increase tissue penetration in your IHC experiments, we recommend you try one of our F(ab')2 fragment antibodies. For non-fluorescent imaging, Abcam also offers a comprehensive range of products for immunohistochemistry. Included in the portfolio are EXPOSE IHC kits, which provide greater sensitivity in comparison to polymer and ABC detection systems.
This is achieved through small detection complexes. Our IHC portfolio also includes the classical biotin/streptavidin kits and reagents. If you would like to know more about our cell imaging products, please visit abcam.com/imaging, where you can find more detailed information by zooming in onto the images. A complication often experienced when using mouse antibodies and mouse tissue is a 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 IgG, ensuring minimal background in addition to a simple and reliable protocol. More about this product can be found at abcam.com/MoM.
Especially for those of you doing IHC, I would like to make you aware that Abcam recently launched the AbTrial programme, which allows risk-free testing of our products. You can take part if you would like to test one of our products, and tested application of species. Species and applications are clearly stated on the individual datasheets. If you would like to take advantage of this offer or simply would like to know more, please go to abcam.com/AbTrial. Abcam's 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. As mentioned in Lucy's introduction, Simon has edited a book on immunohistochemistry which covers many of the topics discussed during this webinar in more detail. So if you would like to get more in-depth information about antigen retrieval methods, selection of reporter labels and automation of your experiments, this is a great resource. The book can be purchased directly via the Abcam website, or on Amazon.
Abcam also organises a variety of conferences during the academic year. Of those, new avenues of brain repair, programming and reprogramming of the central nervous system might be of interest to you. The meeting will take place in June at Harvard University, and more information can be found on our website. The third conference in the Chromatin Replication and Chromosomal Stability series will take place in Copenhagen next June. Keynote speakers are Susan Gasser, Director of the Friedrich Miescher Institute in Basel, Switzerland and Adrian Bird, Buchanan Professor of Genetics at the University of Edinburgh in Scotland. If you are interested in attending, please check out the below link. Also, I would like to highlight our future webinars. We will be delighted if you could join us on January 31st for a webinar on Compensation in Flow Cytometry. This webinar will be presented by Professor Graham Pockley, Associate Director at Nottingham Trent University, and the Head of the Flow Cytometry Facility at Newcastle University, Ian Dimmick.
Also, if you have enjoyed this webinar and would like to learn more about IHC using single and multiple labels, please register for Simon's next webinar on March 6th. To stay with the fluorescence theme, there will be a webinar about fluorescent western blotting on the 28th February. The webinar will be presented by Dr Martin Broadstock from King's College, London. You can listen to all of our webinars after they have taken place by simply going to abcam.com/webinars. To thank you for attending this webinar, we would like to give you a special 25 per cent discount on all secondary antibodies, CytoPainter kits, IHC kits, avidin/streptavidin and RabMAbs. All you have to do to take advantage of this offer, is to quote promotion code ICC-TBAM1 when placing your order. I would like to finish by thanking Simon and all of you for attending, and would like to let you know that we are happy to answer your questions now.
SR: Thank you very much, Judith. So thank you to everyone who has submitted questions for me. I've been reading through them while Judith was speaking and I've pulled out a few of the interesting ones. Firstly, Tom has asked: What is the difference between formaldehyde, paraformaldehyde and formalin? Well, the answer is that they're essentially the same thing, but in different forms. So in order to penetrate cells and adequately fix the proteins therein, aldehyde fixatives need to be monomeric, and formaldehyde is just the monomeric form of the long chain paraformaldehyde which is, incidentally, an insoluble solid at room temperature. So paraformaldehyde is heated in order for it to become monomeric, and therefore soluble in a gaseous phase. But an aqueous solution can only be saturated to 40 per cent with formaldehyde, and this is termed formalin. Diluted working concentrations of this concentrate at 40 per cent formalin, are what is being referred to as formaldehyde.
Another question, so Sarah has enquired: I've found an antibody to the protein that I'm researching and I want to use it in ICC, but ICC isn't a test application on the antibodies datasheet, will it work? Well, the answer is just try it and see, so other test applications can often be useful for gauging this, such as western blot. If there's a single band on the western blot at the correct molecular weight, then there's a high chance that if the antibody does prove successful in IHC or ICC, it will give a very specific positive staining with very little or no background. Flow cytometry is another, since it's very similar to ICC, just a variation on a theme. But it goes without saying that, where possible, you should only purchase an antibody where the datasheet says it's already been successfully characterised in IF or IHC. We have another question from Harjit, who would like to know: What is the best immunogen format for antibodies destined to be used in IHC or ICC? So what I think you're getting at here is immunogen design and format is something completely out of the hands of the end user, unless, of course, you're going to the expense of having a custom antibody produced. But it's certainly something to consider, so you're correct there.
Purified proteins as immunogens tend to generate antibodies with a greater chance of recognising the native protein, rather than a short peptide sequence consisting of up to 20 or so amino acids. Having said that, it's because the proteins - well, this is because proteins exhibit tertiary structure, whereas peptides do not. Recombinant proteins tend to be the next best thing to purify proteins, although they may not exhibit the same degree of tertiary structure, because they are biologically manipulated forms of the protein produced by the non-native host cells, commonly bacteria. What I was trying to get at, is it's not to say that antibodies generate using carefully selected peptides are not of any value, since many are indeed excellent. The characterisation data displayed on a datasheet for a particular antibody, is therefore much more a reliable source of information as to whether or not the antibody will be successful with your chosen application.
Finally, Geneira has asked: With regards to haematoxylin counterstaining, what exactly is bluing? Well, a very good question, yeah. So haematoxylin is acidic in nature, and it is pH and it has a purple colour. However, when you change the pH to alkaline it turns from purple to a pleasing blue colour, which gives better contrast down a microscope. It's simply achieved by placing the slide in running domestic supply water, or tap water as we say here in the UK, or faucet water in the US, whatever you call it. You do that for about five minutes before coverslipping. This water usually has sufficient alkalinity to blue the haematoxylin, but areas where the water hasn't, it can be made so by dissolving about 20 grams of sodium bicarbonate and three and a half grams of magnesium sulphate per litre, and that will give you a nice alkaline water for bluing. Thank you very much for your questions, people, they were excellent.