Immunohistochemistry and Immunoblotting

Immunohistochemistry

Immunohistochemistry has an essential role in the evaluation of muscle biopsies and in examining protein localization. The term ‘protein expression’ is often applied to describe immunohistochemical results, but it should be remembered that the technique only reflects localization of a protein, not the related RNA synthesis, and the gene coding for it may not be active at the time the protein is localized. In addition, absence of labelling may sometimes be because the epitope of the antibody is masked and inaccessible. In addition, RNA from a gene may not be translated into protein, but it is the protein that is of pathological significance. Immunohistochemistry is complementary to histology and histochemistry, and the results should not be interpreted in isolation from other morphological studies and the overall clinical picture. Defects in protein localization may identify an abnormality in the gene encoding that protein (a primary defect) or they may be a secondary response to an abnormality in another gene. Immunohistochemical abnormalities related to a primary defect are of particular importance in assessing recessively inherited conditions, where both alleles are mutated. In several dominant conditions, expression from the normal allele may mask any alteration in the mutant allele and labelling of the normal and mutant protein may be indistinguishable; analysis of secondary defects is then particularly important. In some dominant conditions there may be detectable accumulation of protein. Thus, analysis of both primary and secondary abnormalities has an important role in the assessment of biopsies.

With the rapid advances in molecular medicine the number of primary defects that can be identified with immunohistochemistry is growing, and there is a trend towards classifying disorders according to the protein defect (e.g. dystrophinopathy, sarcoglycanopathy, actinopathy, desminopathy, etc.). In this book, however, we have adhered to the classical clinical classification (see Ch. 8 ), as this still forms the basis for clinical diagnosis. This chapter aims to summarize aspects of methodology for immunohistochemistry and also the analysis of proteins relevant to diagnosis, particularly using commercial antibodies that are readily available to everyone (see ). In the last edition of this book we included a table of useful commercial antibodies and their suppliers, but there are now so many sources to choose from that can easily be accessed on the internet we have dispensed with the table. It is important to remember, however, that different companies market the same clone and titres may vary. In the sections on pathological changes we have retained some historical references to reflect the major contribution of so many workers in the field and updated this with more recent studies and reviews.

Many antibodies are now available for studying diseased muscle, and they have widened our understanding of pathological features, but this chapter is not intended as a comprehensive account of the myriad of features that have been identified by the application of antibodies. The objective of this chapter is to give an overview of methods, the use of immunohistochemistry in relation to defective proteins associated with neuromuscular disorders, and to discuss some of the major muscle proteins that are defective in neuromuscular disorders. There is some inevitable overlap with the chapters relating to specific disorders, and there is repetition of some information, but this is included in order to give a more complete overview. This chapter should be read in conjunction with the appropriate disease-related sections.

Methods for Immunohistochemistry

Immunohistochemistry is used to visualize and localize specific protein components of a tissue. The principle of the technique is the specific affinity of an antibody for its antigen. Allied techniques are the labelling of glycoproteins with lectins, the labelling of receptors with a ligand such as a toxin (e.g. the specific affinity of bungarotoxin for acetylcholine receptors at neuromuscular junctions) and labelling of nucleic acids by in situ hybridization. Similar methods of detection and amplification have been developed for all these techniques, but their diagnostic role is currently less than that of immunohistochemistry.

Use of Automated Machines and Immunolabelling Kits

Several companies now market machines for automated imunolabelling of sections, and their use is now widespread. Although these were originally designed for the use of paraffin, fixed sections protocols can be adjusted to work with frozen sections immunolabelled with brightfield markers. Frozen sections are more vulnerable to the procedures of a machine and may lift off or curl, and consistent results may take time to achieve. In addition, the effect of reagents supplied needs to be considered: for example, low concentrations of the detergent triton X-100 are often a component of the kits supplied and, whilst this can reduce background and give excellent results, it must be remembered that it can affect membrane phospholipids. The immunoglobulin class of the antibody used also has to be considered and methods adapted accordingly. Similarly, the dilution of antibodies and the use of commercial detection kits used for labelling both manually and with a machine (e.g. EnVision or Menarini kits) must be empirically determined. Double labelling of myosin isoforms has been successfully achieved on a machine using brightfield markers (see below; ). Multiplex labelling using more than one antibody and different coloured brightfield chromogens or fluorescent labels are being developed for use on automated machines and may soon have a wider use in routine pathological assessment as multiplex labelling using manual methods is informative ( ). It must be remembered, however, that assessment of each antibody separately may also be necessary as any change in colour in the combined signal will be determined by the proportion of each protein detected. Also, the detection levels of fluorochromes can vary in different channels. Double labelling using fluorochromes in more than one channel is a good control for this. We routinely assess collagen VI and perlecan double labelling using both red and green excitation filters for the visualization of both proteins. When performing double labelling, assessment of each protein individually in serial sections or with two different fluorochromes may then be necessary. Each laboratory must determine for themselves the time/cost and efficiency benefits of using a machine and multiplex methods and be aware that technical issues can arise with any method used. The sections below describe manual methods of immunolabelling that we have found satisfactory over the years and factors to consider.

Tissue and Section Preparation for Immunohistochemistry

As for histochemistry, all tissues should be rapidly frozen in isopentane cooled in liquid nitrogen or propane, and immunolabelling performed on cryostat sections, as described in Chapter 1 . If sections are too thick they may ripple and lift off the slides, a problem that can occur with some antibodies but not others. For immunohistochemistry, we find a thickness of 5–7 μm optimal. The epitope for some antibodies is destroyed or masked by fixation, and unfixed frozen sections are then essential. Some antibodies, however, may only give satisfactory results on fixed material, in which case frozen sections can be post-fixed (see below). This is rare for the antibodies currently used for the diagnosis of neuromuscular disorders. If only formalin-fixed, wax-embedded material is available, various antigen retrieval techniques using pre-treatment of sections with enzymes and/or microwaving can be tried. This is useful for archival material but is not the method of choice for routine studies. In our experience, most antibodies for the diagnosis of neuromuscular disorders give good results on unfixed, untreated cryostat sections. Methanol or acetone or the detergent triton, however, is often used.

As described in Chapter 1 , sections collected on Superfrost or Superfrost Plus slides or uncoated coverslips can be stored until required at −20°C, or lower, if wrapped in clingfilm and/or foil. This has the advantage that batches of biopsies can be labelled in parallel, which is time and cost-effective, and each sample then acts as a control for the others, enabling identification of any technical problems. A batch should never consist of samples all with the same provisional diagnosis (e.g. not all potential cases of Duchenne muscular dystrophy: DMD), and a single biopsy should not be labelled in isolation. If results are needed urgently and a batch of cases cannot be assembled, or if additional or repeat studies are needed, a second sample, preferably of normal muscle, should always be included as a control. Sections should be air-dried at room temperature for about 20–30 minutes before use. If only a few sections from a package of stored frozen sections are required, the remaining sections should not be allowed to thaw as condensation may form and freeze when the sections are returned to the freezer, resulting in artefact when the section is subsequently used.

Fixation of Sections

Many antibodies when applied to muscle sections do not require fixation, and this is our method of choice. Acetone or methanol, however, is frequently used for studies of muscle sections, but in our experience this is often unnecessary for the study of human muscle. Formaldehyde or paraformaldehyde, at various concentrations, is also sometimes used. Acetone or methanol, or permeabilization with Triton X-100 (0.05–0.2%) or saponin is often necessary for localization of intercellular antigens in cultured muscle cells and can also be used on sections. A fixative or permeabilization solution may affect the epitope of an antibody and if fixation is required, the most suitable, and its concentration, has to be determined experimentally.

Immunolabelling and Washing

For manual immunolabelling, dry sections are placed in a moist atmosphere to prevent evaporation of the small volumes of reagents, as described in Chapter 1 . Use of a hydrophobic pen to draw round each section is a convenient way to reduce the volume of reagents used and to prevent reagents spreading. Coverslips or slides can be placed in racks and washed collectively in a dish, or they can be rinsed individually. Three rinses of buffer are required, but the timing of each is variable. It can be from 3 to 5 minutes, or in the case of individual coverslips, 10-second dip washes are adequate. The staining tray shown in Figure 1.10 has a magnetic strip which holds the slides in place and is a convenient way to wash a batch of slides by gently expelling buffer from a ‘squeezy’ bottle over the sections for about 30 seconds. Sections must not be allowed to dry out at any stage or false results and artefact will arise. It is also important to ensure that there are no bubbles in the antibody solution when applied, as no labelling will occur under the bubble.

Blocking Agents

Non-specific background, which reduces the signal-to-noise ratio, may be a problem. Additives to the buffer, such as bovine serum albumin (BSA; 2%) or detergents (0.2% Triton X), or normal serum from the same species as the secondary antibody, can be used to reduce non-specific binding of secondary antibodies but, as with fixatives, they may affect the epitope of the antibody. Some tissue components may contain endogenous enzymes, such as peroxidase or alkaline phosphatase. For example, macrophages contain endogenous peroxidase and blocking agents are needed if cluster of differentiation (CD) markers are localized with peroxidase. Endogenous peroxidase can be blocked with hydrogen peroxide (1%) and endogenous alkaline phosphatase can be blocked with levamisole (1 mM), although the EnVision kit contains a peroxidase blocker. It can sometimes be useful to observe endogenous peroxidase activity and to know macrophages are present without having to use specific cell markers (see Fig. 6.8 ). Endogenous biotin can be blocked by applying unconjugated avidin followed by biotin, available as a commercial kit. We find that there is rarely an advantage in using blocking agents for the study of human muscle; good washing and optimal dilution of the antibodies are more critical factors. Phosphate-buffered saline (0.1 M or 0.2 M) at pH7.2–7.4 is usually used for washing, and commercially available tablets are now a convenient way to make this up.

Primary Antibodies

A wide variety of primary antibodies to muscle proteins are now commercially available, in particular those that detect primary defects in recessive muscular dystrophies. Several companies sell relevant primary antibodies, and most companies now have a website. There are also various directories and hybridoma banks (see Appendix 2 ). Several companies market the same antibody clone, so purchasing an antibody from a different company does not necessarily mean it is a different antibody. Attention should be paid to the characterization of a commercial antibody as this is not always complete on data sheets, and also to the recommended storage of an antibody. If freezing is recommended, an antibody should be divided into aliquots of convenient volume to avoid repeated cycles of freeze–thawing. Company websites (e.g. Abcam) offer useful advice on storage and the length of time that an antibody can be used, which is increasingly important for laboratory accreditation. When possible, characterization of an antibody should be checked in the original publication describing the use of the antibody, or the information obtained from the company. Dilution of a primary antibody should always be assessed by titration in each laboratory, and is dependent on such factors as supplier, poly- or monoclonal antibody, secondary antibody, time and temperature of incubation, and the detection system used. As for washing, phosphate-buffered saline at pH7.2–7.4 is used for diluting antibodies. Incubation times can vary from 30 to 60 minutes to overnight at 4°C. The latter sometimes reduces background and lower dilutions of antibody may be possible. Both monoclonal and polyclonal antibodies are available. The former, released from hybridoma cells, recognize a single epitope and are highly specific. Mouse monoclonal antibodies have been used for many years but rat and rabbit monoclonal antibodies are also now available. Polyclonal antibodies are usually purified from serum following injection of the antigen into an animal species (e.g. rabbit, goat or sheep) and recognize several epitopes on the antigen. A control not using the primary antibody is particularly important when using polyclonal antibodies (see below). Double labelling using primary antibodies raised in different species is relatively straightforward, but if the primary antibodies are both monoclonals from the same species kits are available (e.g. Zenon immunolabelling kits). For double/multiplex labelling it is often possible to use a cocktail of antibodies applied at the same time, provided they do not interfere with each other, and appropriate secondary antibodies then applied for each. If multiple antibodies are applied sequentially, the saturation of the first primary must be considered and any possible binding of the unsaturated first primary antibody with subsequent secondary antibodies used.

Detection Systems

It is common to use an indirect detection method to visualize the primary antibody, rather than directly labelling each primary antibody with a marker, and several commercial kits are now available. Indirect labelling gives more flexibility as it involves applying a secondary antibody against the appropriate immunoglobulin of the species in which the primary antibody was raised, and the same one can then be used to visualize several different primary antibodies. Most antibodies are immunoglobulin G (IgG) immunoglobulins but some are IgM. It is essential to use a secondary antibody to the appropriate immunoglobulin or to use one that recognizes all classes. Secondary antibodies to different IgG classes can be useful for multiplex labelling ( ). The secondary antibody may be directly conjugated to biotin, an enzyme or a fluorochrome. Biotin conjugates are then followed by streptavidin labelled with a marker of choice. This amplification technique has the advantage of enhancing the signal because of the total number of bound avidin molecules. Streptavidin directly labelled with a fluorochrome or a preformed avidin–biotin complex (ABC) may be used. Good enhancement is also achieved with commercial kits (e.g. EnVision or Menarini) and the tryamide system (Zenon immunolabelling technology) in which peroxidase is used to catalyze the deposition of biotin or fluorochrome-labelled tryamide close to the antigen–antibody binding site. The deposited biotin is then visualized with streptavidin conjugated to peroxidase or a fluorochrome.

The choice of label to visualize the antibody is often a matter of personal preference but it is also governed by the type of microscope available, the need for a permanent preparation, the amount and localization of the antigen and the affinity of the antibody used. Enzyme labels, such as peroxidase or alkaline phosphatase, provide permanent results and it is easier to see the overall structure of the tissue, particularly if sections are counterstained after immunolabelling. With fluorescent labels, however, it may be easier to distinguish small areas of localized antibody which appear bright against a dark background, and specificity can easily be checked by changing the excitation filter. Methods for quantifying fluorescent signals are also being developed (see below). Aqueous mountants have now improved which reduce fading, and fluorescence can be retained for several weeks, months or sometimes even years. Good fluorescent nuclear counterstains are also now available (see below). Fluorescent methods are usually quicker to apply and avoid the use of hazardous substances such as diaminobenzidine (DAB). Double and multiplex labelling is easier using fluorochromes and the relationship of different antigens closely localized at the same site is more precisely determined; however, brightfield double labelling is also being used ( ). Illustrative examples of the use of both peroxidase and fluorochromes are given in this book.

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