Seeing Is Believing: Use of Antibodies in Immunocytochemistry and In situ Hybridization

Gloria Hoffman, PhD

Department of Biology, Morgan State University Baltimore, Maryland

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Seeing Is Believing: Use of Antibodies in Immunocytochemistry and In situ Hybridization

The Basics of Titrating Antibodies

The goals of this chapter are to introduce newer approaches for antibody use in immunocytochemistry (ICC) and nonradioactive in situ hybridization (nrISH) and to offer help in understanding how chosen approaches are optimized. We emphasize here information concerning which aspects of the techniques are critical, and which are not, as well as insights into how to troubleshoot these methods when they fail. All the approaches are simply designed to link a molecule in tissue (the antigen) to a visible or fluorescent product via an antibodys ability to serve as an intermediary between the antigen and the colored or fluorescent product. The antigens may be the endogenous molecules of the nervous system, injected tracers, transfected gene products, or molecules attached to probes for ISH in order to localize mRNAs. All these molecules have in common the trait of being capable of generating antibodies when injected into a host species; that is, they are antibody-generating molecules, or antigens. Although many companies now make antibodies against a variety of antigens, knowing how to select an appropriate technique for testing these antibodies in tissue is less obvious. Use of kits for ICC visualization is not always successful, and it is not always obvious who is at faultthe investigator preparing the tissue, the company selling the antibody, or the kit for visualization. When funds are limited, being able to convince a company that their reagents do not work requires that one adequately test antibodies to establish credibility. This section will focus on the optimization of ICC staining protocols, stressing key variations in conditions for conducting ICC tests that influence outcomes. Details of how to perform each of these methods are published elsewhere (Hoffman et al., 1992; Berghorn et al., 1994; Hoffman et al., 2008). The basic steps of ICC techniques are shown below in simplified form. Steps in parentheses are used in some but not all protocols: Fixation of tissue (Embedment of the tissue) Sectioning of tissue block Permeabilizing of cells for antibody access Primary antibody application Application of secondary antibody solution Color deposition (Mounting of sections onto slides) Analysis of stained material

the tissue is fixed and processed. Most investigators will use standard buffered formaldehyde solutions that are either purchased, or made in the lab from paraformaldehyde. While these are effective, for some antigens, fixation will greatly influence the outcome (Hoffman et al., 1992). Two specific conditions are worth highlighting: If the antibody is generated by conjugating the desired protein to a more antigenic molecule, and the link is established by reaction with carbodiimide reactions, then the tissue must be fixed with carbodiimide to expose the epitope that the antibody recognizes. For conjugation that was made with glutaraldehyde, a fixative that contains either glutaraldehyde or another strong dialdehyde may be required as well. Thus, knowing how the antigen was prepared before generating the antibody can be important. There could be additional considerations that influence selection of the fixative. For example, 4% formaldehyde solutions do not deactivate all enzymes, and tissue peroxidase is among the enzymes that remain active. Therefore, if immunoperoxidase methods are selected, measures to block endogenous peroxidase activity are required. If tissue is fixed and targeted for both ICC staining and ISH, whether RNases are still active becomes important. Again, enzyme activity is not eliminated by fixation with 4% formaldehyde but is totally eliminated when acrolein (2.5%) is added to the formaldehyde solution. How the tissue is processed after fixation can also be a factor. Although counterintuitive, mounting sections onto slides before ICC staining requires higher concentrations of primary antibodies for optimal visualization, despite the fact that the sections are often thinner than sections that are processed freefloating. Embedment of the tissue in paraffin may extract the antigen from the tissue, and thus, if sections mounted onto slides are used, fixed frozen material may retain antigenicity better than tissue embedded in paraffin. Use of sections mounted onto slides regardless of whether they had been first embedded can sometimes limit how easily the reagents gain access to the tissue. This feature is especially noticeable when unfixed frozen tissue is placed onto slides and later fixed. Staining for ICC is more difficult with this tissue than would be seen if the tissue were first fixed, and then cut and mounted onto the slides. As Dr. Toth will emphasize in Unraveling Complications, later in this course, some tissue preparation will mask the epitopes that allow antibodies to bind; in fact, a variety of approaches have been devised for restoring epitope availability. In this investigators experience, maximal antigen detection is seen in fixed, free-floating sections; thus, this approach will be emphasized herein. When

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How should tissue be prepared?

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A feature that will influence the range of concentrations that provides optimal antigen detection is how

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tissue samples are small or fragile, the tissue can be embedded in a solution of egg yolk and gelatin, fixed in 4% buffered formaldehyde, cut, and then successfully processed while free-floating. In using egg yolk embedding, one must take care to eliminate contamination with egg white. Postembedment fixation of the blocks is needed and enables the egggel solution to take on characteristics that resemble normal tissue in terms of resilience and stability. It should also be emphasized that free-floating sections can be stored for protracted lengths of time (more than 20 years) in antifreeze solutions at 4C with no loss of antigenicity (Watson et al., 1986; Hoffman and Le, 2004). This measure makes possible the later use of tissue with newly discovered tools without requiring that additional tissue be generated.

good staining for an untested antibody. Of critical importance is that in todays world, antisera can be effective at widely different concentrations: Some antibodies work well at a primary antibody concentration of 1:1,000,000, whereas others give detection at a concentration of 1:1000! Therefore, the most effective test of antibody staining should include the following series: 1:1,000 1:3,000 1:10,000 1:30,000 1:100,000 1:300,000 In other words, antisera should be tested with all other reagents maintained at constant concentrations over a 3-logarithmic (log) scale, in half-log units. Our recommendation is to use the avidinbiotin complex (ABC) method with nickel-enhanced diaminobenzidine (NiDAB) as the chromogen. In this way, variations in fading are not present, and one can make use of some unique features that NiDAB offers to help the investigator to know whether the primary antibody concentration is too high, or too low. Some subsequent adjustment of the concentration between those used for titration may be required after examining the tissue. Not only does the use of ABC methods

Antibody in hand: where to begin?

One first needs an understanding of how antibodies recognize antigens and the steps taken in ICC methods for localizing antigens in the tissue. Typical antibodies are of the IgG class, a cartoon of which is shown in Figure 1. Note there are two domains that convey very different information: The Fc region (stem of the Y-shaped molecules) encodes information about the species of animal that generated the immunoglobulin. Thus, the Fc region is constant for all immunoglobulins the animal makes. The region of the Ig molecule that encodes for the antigen is variable. It contains portions of the long and short chains and is referred to as the Fab domain. Two identical Fab regions bind to antigens. Initially, immunocytochemical studies linked a fluorescent molecule or enzyme directly to the antibody. This approach was only partly successful because often, the antigen binding sites became labeled and began blocking antigenantibody formation. Most methods used today are considered indirect in that the molecule that is visualized or the enzyme is not linked to the antibodies specific for the antigen (termed primary antibodies) but more generally to antibodies generated against the Fc region, termed secondary antibodies (Fig. 1).

Using antibodies for staining tissue

Figure 1. Cartoon showing the structure of an IgG molecule and the features important for use of immunoglobulins for ICC.

Using antibodies for staining tissue requires that the exact concentration for detection be used. Systematic dilution of the primary antibody determines where optimum staining can be obtained. The optimum concentration is influenced by several factors: the method used for detection (covered in the next section), the amount of antigen in the tissue, and the antibodys affinity for the antigen. Note that there is no universal concentration that will always give

with NiDAB provide the most sensitive detection, but the color reaction (when used according to our protocols) enables one to clearly distinguish between primary antibodies that have a high degree of activity but fail to stain because their concentration is too high, and those that have no activity or stain weakly because their antiserum concentration is too low (Hoffman et al., 2008).
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Seeing Is Believing: Use of Antibodies in Immunocytochemistry and In situ Hybridization

One often assumes that if staining is weak, then the primary antibody concentration is too low. This assumption is not true. A feature poorly understood by many investigators is that when too high a concentration of primary antibody is applied, the reaction is quenched, and less product is actually produced. This feature is illustrated in Figure 2 with the titration of an antibody against melanin-concentrating hormone (MCH). Note that at the highest concentration depicted (1:3000), staining is minimal (and uneven). With dilution, however, relatively high background staining becomes more evident despite the appearance of specific staining; as the optimum concentration of primary antibody is approached, the background abates.

The importance of controls: strong staining is not the same as specific staining

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Figure 2. Micrographs depicting a titration of an antibody against melanin-concentrating hormone (MCH).

In the field of ICC, debate continues over what constitutes an appropriate control for antibody reactions. Too commonly, investigators use only the elimination of the primary antibody. Eliminating the primary antibody rarely shows any reactivity unless high levels of endogenous peroxidase activity are present: It provides no information about antibody specificity, but only whether something (but not necessarily the Igs) in the antibody solution was required for staining. The goal of specificity controls is to validate that the tissue has the antigen, and that the antibodies are revealing its correct location. The Journal of Comparative Neurology has required that controls for antibody specificity include either use of a knockout mouse, or presentation of Western blot data to validate staining. While each is an excellent control, neither is universally applicable. Some antisera that are able to stain tissue well in ICC reactions will not bind to the antigen in a Western blot. Conversely, some antisera that are useful for Western blots do not recognize the fixed ligand in tissue. Knockout controls are useful only if the studies are conducted in mice (or the antigen is conserved across species) and the knockout animal is available. Moreover, one has to be careful that the knockout is a gene deletion or minimally, deletion of the sequences encoding for the same portion of the protein formerly contained in the antigen used to generate the antibody. False-positives can be difficult to interpret. In initial ICC studies, the selection of preadsorption with the antigen was considered the best available way of validating antibody specificity. When a negative result after antigen preadsorption is applied, the result suggests that the antibodies are appropriately recognizing the antigen. Positive staining after preadsorption can be used to identify antibody populations to molecules that may have contaminated the immunogen (or were purposely linked to it to increase immunogenicity); it can also identify antibodies the host animal already had when it was immunized (Clayton and Hoffman, 1979). However, care must be taken: If the same antigen used to make the antibody was contaminated with another protein, it will produce the false impression that the antibody is specific. One should use the purified antigen from another source if possible. Also, preadsorption controls do not eliminate staining due to a similar, but not identical, antigen in the tissue that possesses the same or closely related epitopes. For example, the antisera against leucine enkephalin are not able to distinguish staining of methionine enkephalin present in nervous tissue.

Note that NiDAB in titrations also shows a color shift that is useful. When the serum concentrations are too high, the positive staining (when present) has a brown color despite the fact that the NiDAB product should be blue-black. Only when the primary antibody is in the optimal range (1:100,000) does the staining shift to blue-black. The lack of staining at very high antibody concentrations appears as a result of the inability of substrates to reach the enzyme. For immunofluorescence detection, the quenching of staining when primary antibody concentrations are too high can also be observed; however, it can be more difficult to distinguish low reactivity when the antibody concentrations are too high compared with those produced by nonspecific staining when the antibody simply does not bind or is too dilute. The exact concentrations that are too high vary with each antibody preparation, and this is another reason that titrations are so critical. With certain fluorescence methods, eliminating background is difficult.
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In some circumstances, the antibodies are specific, but a simple preadsorption test fails. For example, when the antigen has been modified by a chemical reaction to link it to a larger protein to generate the antibody, then a similar chemical modification may be necessary to enable antibodies to bind to the antigen. To illustrate, for staining of histamine or serotonin in tissue, the nascent amine is not able to bind to the antibodies. Histamine antisera are generated by reaction with carbodiimide; serotonin antibodies are generated through reaction of the monoamine with formaldehyde. Thus, antisera against histamine will be capable of recognizing only histamine that was complexed to a protein in the presence of carbodiimide, and serotonin must similarly be converted to block the staining. Preadsorption should be conducted with the same altered molecule.

will dictate a change in the optimum concentration of the primary antibody. These differences are predictable once a standard ABC/NiDAB titration has been conducted. For the reasons cited in the previous section, The Basics of Titrating Antibodies, I recommend using the ABC immunoperoxidase method with NiDAB as the chromogen for titrating antibodies.

A note about methods and their nomenclature

eliminating background staining

There are many descriptions of additives that reduce nonspecific binding of ICC reactants and thereby lower background staining. Knowing a priori whether any one will work is not always possible. There are some exceptions. If protein conjugates were used in generating the antibody, there will be a need to add the proteins used to increase antigenicity (thyroglobulins, serum albumins, or limpet hemocyanin), though other additives may not be necessary. Most frequently, investigators add a 1% solution of normal serum from the species used for generating the secondary antibodies. In general, we recommend not adding anything unless the background staining is high. An exception is when tissue is fixed by immersion or is poorly perfused and has a great deal of residual blood. Red blood cells contain high levels of peroxidase, and this activity needs to be blocked when employing ICC reactions that use peroxidase reactions for detection. Red blood cells will also present a problem for fluorescence if a red fluorophore is selected, since hemoglobin fluoresces red. A question that is frequently asked is how much effort should be made to rescue poor staining. Procedures to reduce background labeling can be effective, but if an antibody is poor for antigen detection, no amount of treatment will make the procedure work well.

The most common indirect ICC methods used today are classified as those that are immunofluorescent (linking the antibody complex to fluorescent molecules) and those whose products are generated by enzyme reactions and are visible under a light microscope. The latter class uses either horseradish peroxidase or alkaline phosphatase reactions; a range of substrates for each enzyme provides different colored products in the visible range, some of which are also fluorescent. Several newer methods comprise a hybrid of the two approaches and generate fluorescent products through enzyme reactions, as will be discussed in subsequent chapters herein by Drs. Roth and Toth (Tyramide Signal Amplification Strategies for Fluorescence Labeling, and Unraveling Complications, respectively). The various methods are cartooned below in ascending order of complexity/sensitivity.

Simple, indirect methods: primary antibody + fluorophore/enzyme conjugated to the secondary antibody

This method is the least sensitive among commonly applied immunofluorescence methods, yet is the most commonly used (Fig. 3A). Only a limited number of molecules can be linked to the secondary antibody. To ensure that sufficient numbers of tagged molecules bind in the antibody complex, a higher

How the Methods Differ in Sensitivity

Which method to use?
A survey of the literature will reveal many methods available for ICC studies. Which one to choose is often based on the type of microscope available to the investigator and the time needed to perform the studies. As will be apparent from the remainder of this section, differences in the methods sensitivity (defined as the amount of product generated by the antigen)

Figure 3. Cartoons of the ICC staining using secondary antibodies that are either labeled with a fluorophore (A), or labeled with an enzyme (B) whose product after reaction with a substrate is colored.
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Seeing Is Believing: Use of Antibodies in Immunocytochemistry and In situ Hybridization

concentration of primary antibody is required than would be needed for more sensitive methods. The enzyme-tagged approach provides greater sensitivity than the fluorescent variant (Figs. 3B, 3C). A difference can be noted between alkaline phosphatase and peroxidase: For some alkaline phosphatase products, as the time of reaction is lengthened, more and more product is formed. With the most common substrate, nitro-blue tetrozolium, the cell can become so filled with product that it loses compartmental distinctions. In contrast, the common peroxidase substrate diaminobenzidine (reacted either in the presence or absence of nickel salts) forms an insoluble precipitate product. This product will eventually cover the enzyme and antigenic sites on the primary or secondary antibodies, limiting the further generation of product while retaining the highest resolution. This feature is of great use in ISH reactions, as will be discussed subsequently in Nonradioactive In situ Hybridization.

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ABC: primary antibody + biotinylated secondary + streptavidin-fluorophore / streptavidin-enzyme

Figure 4. Cartoon of ICC staining with avidin-biotin complexes (ABCs) using a fluorescent avidin (streptavidin fluorophore).

This method offers greater numbers of molecules for detection than are present in the previous approach, since for each biotin molecule present on the secondary IgG, four molecules of avidin can bind. As a result, there is about a threefold increase in the amount of fluorescent signal for each molecule of bound primary antibody compared with that obtained with the analogous method using a direct tagged secondary antibody (Fig. 4). Translating this feature to determine an optimal primary antibody concentration, threefold less primary antibody is needed; and, since more signal is present, the amount of product generated is greater. Thus, the time of exposure for fluorescence needed to photograph the specimen is shorter, and for either fluorescence or enzymatic detection, the signal-to-noise ratio is higher. The enzymatic variant of this approach (Fig. 5), in which a biotinylated enzyme is linked into the system through an avidin link, is even more sensitive since enzyme product further amplifies the signal. This approach enables 50-100 less primary antibody than the directly tagged secondary methods. ABC peroxidase is now the most commonly used of the immunoperoxidase methods.

Figure 5. Cartoon of ABC ICC staining using an enzyme (E) link in the complex.

Tyramine signal amplification with biotinylated tyramine as a substrate

This method combines primary antibody + (biotinylated secondary + avidin peroxidase) and biotinylated tyramine (with peroxide) as the substrate, followed by a streptavidin fluorophore/enzyme incubation (Fig. 6). It uses an increase in signal due
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Figure 6. Cartoon depicting amplification of the ABC complex through use of a tyramine signal amplification (TSA). Visualization is accomplished with a streptavidin fluorophore.

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to the generation of multiple biotin molecules at the site of the antigen through an ABC enzymatic reaction involving biotinylated tyramide (a substrate for peroxidase that, with peroxide, enables multiple molecules of biotin to be deposited at the site of the antibody complex). Incubating the tissue with streptavidin linked to a fluorophore completes the reaction. The result is a highly amplified fluorescent signal (Berghorn et al., 1994). The fluorescent product is bright, and the sensitivity of the overall reaction is far greater than that produced by any of the other fluorescence methods. Primary antibody concentrations to obtain optimal product are 100-200 lower than would be needed for the directly tagged, secondary methods. As will be discussed in a subsequent chapter (Tyramide Signal Amplification Strategies for Fluorescence Labeling), the overall reaction can be further stimulated by using buffers with high salt concentrations. It can be used with peroxidase diaminobenzidine (DAB) detection, but since it becomes so sensitive, it is quite difficult to use owing to the crowding of complex components (as discussed in The Basics of Titrating Antibodies, above).

tissue preparation. Using sections that are stained while freely floating gives reagents optimal access to the tissue and allows excess reagents to be effectively rinsed away, giving products for detection the highest sensitivity and signal-to-noise ratios.

Selecting the right combination of methods

Before beginning to stain tissue for two different molecules, one must evaluate each separately and determine whether the two molecules are likely to be found in the same or different structures within the tissue. The options for double-labeling are different if both molecules are within the same cells and in the same cellular compartment in approximately the same amounts, rather than in different compartments within the same cells or in two different cells. Whether the two antibodies used to stain the tissue are generated in the same or different species also can influence which methods can be combined. Before outlining double-labeling strategies, it is important to recognize that each method for ICC offers a number of advantages and disadvantages. Combinations for double-labeling will use the following approaches: (1) The ABC approach detected with the peroxidase substrate DAB, or DAB reacted in the presence of nickel salts (NiDAB). (2) The ABC approach detected with a streptavidin fluorophore. (3) Immunofluorescence using biotinylated TSA with detection via strepavidin linked to fluorophores (Berghorn et al., 1994; Hoffman et al., 2008). (4) Indirect staining with a secondary antibody linked to alkaline phosphatase, followed by reaction with fluorescent substrates for the enzyme (McDonald et al., 2000). (5) Indirect immunofluorescence using secondary antibodies prejoined to a fluorescent molecule.

General recommendations

For all the methods, the incubation time with the primary antibody is 24-48 hrs at 4C. Incubation with secondary antibodies and subsequent reagents are applied as detailed in our recommended protocols (Hoffman et al., 2008). The most important take-home message when selecting a method for staining is that the concentration of primary antibody needed to obtain optimal localization is determined by the sensitivity of the method. Thus, the same dilution of primary antibody does not result in optimal staining across methods. Although one often is given a recommended antibody concentration for ICC by a company, unless the method used for screening is specified in detail and includes the conditions used for preparing the tissue and performing the assay, the suggested concentration can be orders of magnitude different from what would provide reliable staining in your experiment.

Selecting Methods for DoubleConsiderations of which methods to Labeling

This section will cover some of the basic strategies for double-labeling, how to select the right combinations of methods, and how each of the methods is performed. Although the results presented here are based on use of tissue that is processed free-floating, with some adjustment they can be applied to any

combine

A decision of which methods will be best for double-labeling requires understanding, most importantly, where within the tissue the two antigens are located. After examining each antigen, the choices are as follows.
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Seeing Is Believing: Use of Antibodies in Immunocytochemistry and In situ Hybridization

Antigens present in two different cells or two separate cellular compartments in the same cell ABC peroxidase (NiDAB) + ABC (DAB) ABC peroxidase (DAB or NiDAB) + any fluorescence method Alkaline phosphataselinked secondary antibodies (fluorescence detection) + any other fluorescence method only when primaries are generated in different species Direct-tagged secondary antibodies for both only when primaries are generated in different species TSA-amplified fluorescence + fluorescencetagged secondary ABC fluorescence + TSA amplified fluorescence two methods using biotin cannot be combined Staining tissue to reveal the labeling of two antigens present in different cells, or in distinctly different compartments in the same cell, enables the greatest flexibility for selecting a method. One could easily use two different immunoperoxidase reactions (i.e., NiDAB and DAB; Fig. 7); one immunoperoxidase step and one of the immunofluorescence methods (Fig. 8); or selected double-immunofluorescent approaches (Figs 9, 10, and 11). In considering approaches that use immunoperoxidase reactions with DAB as a substrate (with or without nickel salts), it is important to recognize that, as the oxidized substrate is deposited in the tissue, the DAB product is insoluble and thus does not diffuse from the site of the enzyme. Eventually, the DAB product will cover the sites where the enzyme is located and prevent any further reaction from occurring. Since the enzyme lies over the antigenantibody complexes, these also will be masked by the precipitate. This feature limits the spread of product but, more important, prevents further reactions with any of the complex components. A second application of secondary antibody will not bind to the reacted primary antibody; thus, it is possible to use sequential reactions with two ABC peroxidase methods even if the two primaries were generated in the same species. A variant of this method uses one of the fluorescent strategies along with ABC immunoperoxidase labeling (Fig. 8). The ABC method is performed first, followed by the immunofluorescence method. When NiDAB is used in this type of protocol, any fluorescent molecule will be useful. If DAB rather than NiDAB is used, fluorophores that emit in the
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Figure 7. Double-labeling strategy that uses two reactions of peroxidase (P) for antigen detection; one with NiDAB as the substrate (black) and the other with DAB as the substrate (brown). Provided the NiDAB reaction is run to completion, the brown and black stains do not mix.

Figure 8. Double-labeling that employs the ABC reaction for the first antigen followed by fluorescence with a tagged secondary antibody for the second.

red wavelengths ~600 nm cannot be used, but those that emit at lower wavelengths can. If dual fluorescence methods are selected, the most important consideration will be whether the two primary antibodies were generated in the same or different species. If primary antibodies were generated in different species, then alkaline phosphatase-linked secondaries can also be used for one, and either biotin-based immunofluorescence (TSA-amplified or standard ABC) or direct fluorophore-tagged secondary antibodies can be applied for the second (Fig. 9). Two fluorescent-tagged secondary antibodies can also be applied (Fig. 10). In addition, it is possible to combine TSA amplification with fluorescence using a fluorescent-tagged secondary (Fig. 11). Which of these fluorescence methods will work best depends on a number of factors that include the fol-

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lowing: (1) the amount of antigen present (if very different, then it is best to amplify the least abundant of the two antigens to better equalize their detection) and (2) the relative volumes of each antigen in the cell compartment. If one antigen occupies a very small subcompartment, it is best detected if TSA amplification is applied. Amplification may also be helpful if the signal-tonoise ratio of one of the antigens is low while using fluorescence with a direct-tagged secondary. Under these conditions, amplification will better separate background staining from specific labeling. Our recommendation is that TSA amplification be used for one of the two antigens unless both are present in high abundance. If the two primary antisera are generated in the same species, TSA amplification can be combined with a method that uses a directly conjugated secondary antibody (Shindler and Roth, 1996). Biotin-based approaches in double immunofluorescence techniques can only be used once, however. Antigens present in the same cells and in the same cellular compartment ABC peroxidase (NiDAB) + ABC (DAB) The two products are not easily distinguished in the same compartment ABC peroxidase + any fluorescence method The opacity of the DAB product obscures fluorescence detection TSA-amplified fluorescence + fluorescencetagged secondary Alkaline phosphataselinked secondary antibodies (fluorescence detection) + any other fluorescence method only when primaries are generated in different species Direct-tagged secondary antibodies for both only when primaries are generated in different species and detection is strong ABC fluorescence + TSA amplified fluorescence Avidin-biotin interactions make separating two products impossible
Figure 11. TSA amplification of the product of the first antigen/ antibody series followed by use of a direct fluorophore-tagged secondary for the second provides increased sensitivity of the detection of the first antigen.

Figure 9. Double-labeling that uses alkaline phosphatase tagged secondary antibodies for one antigen, and fluorophorelinked secondary antibodies for the second.

Figure 10. Double-labeling using two fluorophore-tagged secondary antibodies.

When two antigens are expected in the same cellular compartment in approximately equal amounts, the choices for staining strategies are more limited than when the two antigens are found in separate cells or
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in very different compartments. Only fluorescence methods can effectively distinguish the two antigens within the same compartments. In using two fluorescent methods, one must choose a way to achieve sufficient sensitivity from the method in order to see each antigen. Should one series of reactants be amplified? Are the two antibodies generated in the same species? Is one antigen much more abundant than the other? In addition, which fluorophore should be used for which antigen needs to be considered. Can the microscope used discriminate the two fluorophores? Without very sophisticated equipment, eliminating the excitation range of the first fluorophore that overlaps with that of the second fluorophore can produce false-positives. False-positives can result if using one antigen that is in much higher abundance than another, or one that is amplified versus one that is not. This will take place if a green-fluorescent fluorophore is applied to the reaction with the highest abundance and coupled with a standard red fluorophore. To see why this occurs requires an understanding of the spectral characteristics of each fluorescent dye (shown in Fig. 12, for Alexa dyes 488 and 548two of the commonly used green and red fluorophores). The same issue pertains to fluorescein and rhodamine, or for Cy2 and Texas Red fluorophores, or for some of the newer dyes such as DyLight molecules [Thermo Scientific, Rockville, IL]). When both fluorophores are present in equal amounts, the two colors remain separate. Very little green fluorophore is excited by the wavelengths that excite the red fluorescent molecule, despite the fact that a slight tail in the excitation spectrum is found in the range where red fluorescence is excited (Fig. 12A). If, however, the amount of the green fluorescent molecules is now greatly increased over that of the red fluorescent one, a considerable amount of green fluorophore extends into the wavelengths that enable emission of fluorescence in the red range (shaded areas Fig. 12B). As a result, with filters in place for visualizing the red fluorescence, the green fluorophore will appear red. When it is the red fluorophore that is amplified (Fig. 12C), no greater bleed through of the green fluorophore is found, keeping the fluorescence of the two molecules optically separate. Thus, investigators are urged to use fluorescent molecules that emit in the red wavelengths for amplified or highly abundant signals. One can determine ahead of time whether bleedthrough will be a problem. A critical control step before proceeding to put two fluorescent molecules together is to take each reaction alone and to validate that the fluorescence can be generated only by the appropriate filter combination.
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Successful antigen localization in double-labeling requires understanding that different methods require different primary antibody concentrations to achieve optimal staining

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As was mentioned in How the Methods Differ in Sensitivity, above, there are differences among the various methods with respect to how much primary antibody is required for optimal labeling. If the choice of primary antibody for double-labeling is not in the most effective range, unexpected and confusing results can be obtained. To illustrate this point, we present the following scenario. Two antisera are used in a study to determine the axon innervation pattern of transmitter #1 onto neurons expressing transmitter #2. ABC immunoperoxidase with NiDAB detection was used for transmitter #1 in the axons. DAB staining with an ABC reaction was used for transmitter #2 to reveal the neurons being innervated. Single-labeling

Figure 12. A, Absorbance (excitation) spectra and emission spectra highlighting the patterns that would be seen using a standard fluorescence microscope. The boxed areas show the bandwidth of common filters for red fluorescence. Note that there is very little absorbance from the green fluorophore with use of the red filters. Thus, the green fluorophore would not result in substantial red signal.

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Figure 12. B, If the amount of green fluorophore generated in the reaction is greatly increased, then a substantial amount of fluorescence from the green fluorophore absorbs light in the red range (gray area on left). This would be seen as red fluorescence (gray area on right) and false-positive double-labeling results. The section had only been reacted with green fluorophore, yet it appears to be fluorescing both green and red.

Figure 12. C, When the amplification reaction for immunofluorescence is reversed (amplifying the red fluorophore), no falsepositives are found, since no appreciable absorbance/emission of the green fluorophore in the range of the excitation/emission filters is found.

using each method generated acceptable results (Figs. 13A, 13B), but for the one using NiDAB, the time of staining was shortened to avoid back-

ground staining. When the double-staining reaction was performed, the results were striking (Fig. 13C). No evidence of the second (brown) antigen was
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found, and the background in the tissue turned dark brown. With this result, it was tempting to conclude that something had gone wrong with the staining of the second antigen with DAB, when in fact it was the staining of the first antigen that caused all the problems. When using an antibody at a concentration that is too high but employing a staining time that is too short to complete the reaction of deposited peroxidase, staining will finish upon reexposure to DAB. Brown-black mixing will occur, and often, no product from the second antibody complex will be found. Diluting the first primary antibody solves the problem (Fig. 13D). Similar considerations must be applied if fluorescence methods are used and antisera concentrations are too high for optimal detection. This feature suggests that crowding of antibodies or the complexes that label them is responsible for the discoloration.

advanced our understanding of the composition and function of the nervous system. Techniques for measuring levels of RNA using real-time (rt) PCR and quantitative rtPCR have made it possible to assess how gene expression changes under physiological and pathological conditions. This, too, has greatly added to our knowledge of the nervous systems function and plasticity. Yet it must be appreciated that the nervous system is a highly diverse organ system. Eventually, one must ask where detected genes are expressed, and whether observed changes are seen in all, or only in select, populations of cells. It is within this realm that ISH strategies provide an effective means for determining where genes are expressed. Two basic approaches are used for ISH: isotopic (radioactive) methods and nonisotopic (nonradioactive) approaches. Isotopic ISH methods employ nucleotide sequences complementary to the cells RNA template that have an incorporated radioactive signal (3H, 35S, 32P) on one of the nucleotides. When reacted (hybridized) with the tissue, the label is detected by exposing film or emulsion to the section in order to reveal the radioactive label. Most quantitative assessments with ISH use this approach.

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Nonradioactive In situ Hybridization: A New Twist on an Old Theme

The characterization of the genomic codes for humans, mice, rats, and a variety of other species has

Figure 13. Proper antibody titrations are important for double labeling. A, Shortened staining times for the first of two sequential antibody/antigen series may produce good staining. B, Verification of the second antigen/antibody staining series alone also shows clear and high quality labeling. C, When combined, no clear double labeling is found; high brown background is present. D, When the first primary is properly diluted, then clear double labeling is possible.
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One feature of isotopic ISH is that the detected signal is found at some distance from the labeled nucleotide. As a result, isotopic methods often lack precise cellular resolution of the signal: Invariably, there is spread of the signal as it travels from the tissue to the emulsion or film. In addition, there can be some decay in the strength of the signal (depending on the isotope) that worsens as a function of the distance between the labeled nucleotide and the emulsion. Thus, with standard 20 m sections, only the part of the section closest to the surface is accurately monitored. Nonisotopic or nr-ISH avoids problems of signal spread or decay by enabling the label to be detected within the tissue at the site of the hybridized sequence. Two labels are commonly used for light microscopic nr-ISH: digoxigenin and biotin. Both are small, planar molecules and are easily stained using antibodies against these molecules. For digoxigenin, antibodies that are either unlabeled, or are conjugated to alkaline phosphatase, are commercially available. The latter have been employed in most studies with digoxigenin. Use of digoxigenin in nr-ISH labeling is presented in detail in the chapter In situ Hybridization Using Antibody-Based Methods by Dr. Baskin. It should be apparent that use of either digoxigenin or biotin-tagged nucleotides, followed by ICC detection, can be viewed as a simple modification of standard ICC; many of the same approaches that are useful for ICC will be useful for ISH. Indeed, our group and others have tested this hypothesis directly after realizing that, just as ICC detection of molecules is greatly improved by use of freely floating tissue, so is ISH.

Fluorescein-labeled nucleotides One approach that is theoretically simple is the use of a fluorescent molecule tagged to a nucleotide. One of these, fluorescein, is sometimes used for detecting DNA sequences in cells. For nr-ISH, our experience indicates that no fluorescent signal can be obtained from the fluorescent tag, but when ICC is applied using an anti-fluorescein antibody, followed by ABC detection of the antibody complex, a clear signal can be obtained (Fig. 16).

Biotin-labeled nucleotides for ISH

Many years ago, scientists stood up at a meeting and declared that nr-ISH with biotin-labeled nucleotides was associated with too high a level of background

Figure 14. Cartoon of nr-ISH that uses digoxigenin-tagged nucleotides (black triangle) and antibodies against digoxigenin that are conjugated to alkaline phosphatase (AP). The most common substrate for the enzyme reaction is nitrotetrozolium blue (NTB), and an example is shown in the micrograph insert.

Use of digoxigenin-tagged nucleotides in free-floating sections processed for ISH

As shown in Figure 14, digoxigenin-labeled nucleotides that are subsequently stained using alkaline phosphataselinked anti-digoxigenin and nitrotetrozolium blue as the substrate provide strong, clear staining signals in tissue. Background labeling is much lower than is usually found with isotopic methods. Details of the basic method have been published (Elias et al., 1998).

Peroxidase-labeled anti-digoxigenin

A variant of the enzyme-linked nr-ISH employs peroxidase linked to the anti-digoxigenin. As Figure 15 shows, this method also provides a clear signal of the probe.

Figure 15. Variant of the nr-ISH with digoxigenin-labeled nucleotides that use anti-digoxigenin antibodies conjugated with peroxidase (P). NiDAB staining of the peroxidase is shown in the micrograph insert.

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Figure 16. Nucleotides conjugated to fluorescein (green triangles) may not provide sufficient fluorescence alone, but antifluorescein antibodies enable ICC detection.

labeling, low signals, and was unable to be used reliably. Of course, that statement has not been proven, and may have been the result of a lack of understanding of how ICC should be optimized. For biotinlabeled nucleotides in ISH protocols, a wide range of approaches can be used for detection. These can take many forms: They can be simple (as would be obtained with a streptavidin-fluorophore); they can employ the linking of biotin to either avidin-peroxidase or avidin alkaline phosphatase; or they can be made more complex through use of anti-biotin followed by ABC methods (Fig. 17) or ABC+TSA amplification (Fig. 18).

Figure 18. TSA amplification of the ABC reactions enables increased fluorescence signals for the ni-ISH reaction (Cy3-strepavidin was used for the fluorophore).

ize the label requires that the antibody be properly titrated. We have learned that if antisera against the tags on the nucleotides are used under optimal conditions, reliable quantitative data are obtained. With use of freely floating fixed tissue, some steps required for slide-mounted tissue become unnecessaryin particular, prehybridization or blocking steps. Instead, one need only perform the following simple steps: Prepare probe Prepare tissue. ybridize with probe (temperatures may need H to be reduced to about ~50C so that tissue is not cookedknow that your body hybridizes nucleotides at 37C) Posthybridization washes Develop label (ICC starts here)

Figure 17. Nucleotides conjugated to biotin enable ABC peroxidase detection with anti-biotin antibodies. Either DAB or NiDAB is useful for the enzymatic reactions.

Our laboratory has developed and tested biotin-based ISH approaches (Hoffman et al., 1995; Berghorn et al., 2001; Hoffman and Le, 2004; Koban et al., 2006; Smith et al., 2006; Koban et al., 2008). The remainder of this chapter will illustrate methods for detecting biotinylated nucleotides. For this approach, some of the same principles outlined for ICC apply to ISH. Key among them is that the use of antisera to local 2008 Hoffman

Tissue preparation We have discovered that tissue that is fixed with 4% paraformaldehyde:2.5% acrolein and then stored in antifreeze cryoprotectant well maintains both the antigenic proteins and mRNA (Hoffman and Le, 2004). The critical feature of the latter is that acrolein appears to destroy the activity of ribonucleases (RNases) in the tissue. Acrolein fixation also makes the thin sections (25 m) more resilient. Probe selection Our experience indicates that cDNA or riboprobes that are between 350 and 800 bases in length are optimal for generating enough signal to detect, and

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for enabling good probe penetration into the tissue. Earlier studies by Trembleau and Bloom (1995) applied a similar approach by using biotinylated oligonucleotide detection and examination at both the light- and electron-microscope level. Their data showed that the cytoplasmic mRNA clusters accurately reflected expected compartmentalization of the mRNA, with localization at the endoplasmic reticulum, though not the mitochondria or Golgi apparatus. Probe sizes are optimal at 300-800 bp. Verylow-abundant sequences may be difficult to detect, and for those, radioactive ISH may be the best tool. Hybridization In our experience, biotinylated UTP works best for nr-ISH. Working with multiple probes used for biotin-based ISH indicated that concentrations of the probe in the range of 600-800 ng/kb/ml provide reliable mRNA detection. A range of temperatures for hybridization from 37C to 60C revealed that a maximum temperature of 50C maintained probe specificity without damaging the floating sections. Protein antigenicity is also well maintained through this procedure. Following rinsing and treatment with buffers to remove unreacted probe, the tissue is ready to begin ICC detection. Probe detection In theory, detecting the label on the probe is no different for ISH than for ICC. Our standard strategy is to obtain an antibody against the label, titrate it as we recommended for ICC, and then use the optimal concentration for batch staining. As with other ICC reactions, incubation with the primary antibody lasts 48 hrs at 4C. ICC of biotin in ISH using ABC peroxidase staining has some key differences from the routine ICC method, since the level of background staining has to be kept extremely minimal in order to see the fine, small RNA clusters in the tissue. The secondary antibody concentration is lowered, the ABC reactant concentrations are lowered, and the NiDAB/H2O2 concentrations are reduced as well. Titration of the antibodies in nr-ISH is important, whether digoxigenin or biotin tags are used (Fig. 19). When anti-digoxigenin is too concentrated, background labeling is increased. Quantification of nr-ISH signals Enzymatic detection of immunoreactive products can be useful for determining the level of RNA present in tissue. For ABC methods using peroxidase detection, this feature is reinforced by the fact that the product masks the enzyme sites, limiting further generation of product. As a result, the amount of product will accurately reflect the amount of incorporated label. This feature is shown in Figure 20, which depicts

Figure 19. As with any of the ICC methods, a proper titration of the anti-tag antibody (shown for anti-fluorescein) is required to optimize the assay.

Figure 20. Nonradioactive ISH using biotinylated riboprobes enables useful and accurate quantitative assessment of mRNA levels. A) a section through the hypothalamic paraventricular nucleus from a control rat reacted for vasopressin mRNA. B) Section through the same region from a rat that was hyponatremic for 7 days shows markedly suppressed mRNA expression.

vasopressin mRNA under conditions where the gene is expressed normally or suppressed after the animals become hyponatremic (Hoffman et al., 1995). The ISH signal clearly reflects the level of mRNA, and quantification of the nr-mRNA clusters provides a reliable estimation of mRNA expression (Berghorn et al., 2001; Koban et al., 2006; Koban et al., 2008).

References

Berghorn KA, Bonnett JH, Hoffman GE (1994) cFos immunoreactivity is enhanced with biotin amplification. J Histochem Cytochem 42:1635-1642. Berghorn KA, Le WW, Sherman TG, Hoffman GE (2001) Suckling stimulus suppresses messenger RNA for tyrosine hydroxylase in arcuate neurons during lactation. J Comp Neurol 438:423-432.
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Clayton CJ, Hoffman GE (1979) Immunocytochemical evidence for anti-LHRH and anti-ACTH activity in the F antiserum. Am J Anat 155:139-145. Elias CF, Saper CB, Maratos-Flier E, Tritos NA, Lee C, Kelly J, Tatro JB, Hoffman GE, Ollmann MM, Barsh GS, Sakurai T, Yanagisawa M, Elmquist JK (1998) Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J Comp Neurol 402:442-459. Hoffman GE, Smith MS, Fitzsimmons MD (1992). Detecting steroidal effects on immediate early gene expression in the hypothalamus. Neuroprotocols 1:52-66. Hoffman G, Berghorn K, Knapp L, Le W, Sherman T (1995) Physiological stimulation of vasopressin and oxytocin neurons: perspectives from Fos activation. In: Neurohypophysis: Recent progress of vasopressin and oxytocin research (Saito T, Kurokawa K, Yoshida S, eds), pp 151-164. Amsterdam: Elsevier Science. Hoffman GE, Le WW (2004) Just cool it! Cryoprotectant anti-freeze in immunocytochemistry and in situ hybridization. Peptides 25:425-431. Hoffman GE, Le WW, Sita LV (2008) The importance of titrating antibodies for immunocytochemical methods. Curr Protoc Neurosci 2.12 (in press). Koban M, Le WW, Hoffman GE (2006) Changes in hypothalamic corticotropin-releasing hormone, neuropeptide Y, and proopiomelanocortin gene expression during chronic rapid eye movement sleep deprivation of rats. Endocrinology 147:421-431. Koban M, Sita LV, Le WW, Hoffman GE (2008) Sleep deprivation of rats: the hyperphagic response is real. Sleep 31:927-933. McDonald TJ, Le WW, Hoffman GE (2000) Brainstem catecholaminergic neurons activated by hypoxemia express GR and are coordinately activated with fetal sheep hypothalamic paraventricular CRH neurons. Brain Res 885:70-78. Shindler KS, Roth KA (1996) Double immunofluorescent staining using two unconjugated primary antisera raised in the same species. J Histochem Cytochem 44:1331-1335. Smith JT, Popa SM, Clifton DK, Hoffman GE, Steiner RA (2006). Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J Neurosci 26:6687-6694.

Trembleau A, Bloom FE (1995) Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous non-radioactive oligodeoxynucleotide probes. J Histochem Cytochem 43:829-841. Watson RE, Wiegand SJ, Clough RW, Hoffman GE (1986). Use of cryoprotectant to maintain longterm peptide immunoreactivity and tissue morphology. Peptides 7:155-159.

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