Immunohistochemical Markers for the Rodent Immune System

Abstract

The responses to insults including chemical toxins, irradiation and infectious agents involve morphologic, biochemical and molecular changes in the immune system. The changes in specific tissues and cells often can be detected by histopathology and its associated field of immunohistochemisty (IHC). Cells normally express specific proteins (antigens) that can be detected by IHC. When responses to xenobiotics occur, cells often up or down regulate proteins. The art of IHC requires specialized procedures for detection of antigens. Fixation, tissue processing, immunoreactions and antigen retrieval methods are important elements of IHC. We review the antibodies, their sources, use of frozen or fixed paraffin-embedded tissues and specific IHC methods including antigen retrieval and illustrate how they can be effectively used to characterize the immunotoxicologic effects of agents.

Introduction

Exposure of rodents to exogenous agents including toxins and infectious agents often involves responses in the immune system. Toxicity in the immune system may include degenerative, necrotizing, proliferative and neoplastic responses. The nature and extent of the responses may be agent and dose related. The responses are often measured with biochemical, molecular, cytological, and histopathological methods. Histopathology can demonstrate morphologic changes in normal anatomical and histological structure of thymus, lymph nodes, spleen, Peyer’s patches, and lymphoid cells and tissues in other organs. Gene expression, as detected by antigens in cells and tissues by immunohistochemical (IHC) methods, can provide an excellent adjunct to routine histopathology. Normal distribution of cells expressing specific antigens can be studied with frozen and paraffin sections with a variety of antibodies and IHC methods. The changes in cells and tissues can be assessed and immunotoxicity characterized. This review describes IHC techniques the authors have used in 4 different research laboratories over the past years. We include screening methods for basic markers of the immune cells as well as more detailed methods of analysis.

Technical Considerations

The fixative, specific antibodies, sources of the antibodies, and IHC methods play an important role in IHC. Many aspects of these important considerations have been reviewed in several excellent books and reviews (Romas-Vara et al., 2005). We will not review these considerations here except for some that apply to rodents.

Sources of Antibodies for Rat and Mouse IHC

Major commercial sources of antibodies against specific rodent antigens include BD Biosciences, Serotec, and Santa Cruz Biotechnology, but many other sources also exist. If antigens, such as CD3 and immunoglobulin kappa light chains, are well conserved in many species, anti-human antibodies work well in mice. Frozen sections are the optimal material to use and hundreds of antibodies against rodent antigens work well with frozen sections (BD Biosciences Mouse CD Chart 〈http://www.bdbiosciences.com/pdfs/other/01-81004-36.pdf〉, BD Biosciences Rat CD Chart 〈http://www.bdbiosciences.com/pdfs/other/03-8100095-11A.pdf〉, and a subset work well with paraffin embedded tissues (Cattoretti and Fei, 2000; van den Berg et al. 2001a, 2001b; G. Cattoretti, 〈http://icg.cpmc.columbia.edu/cattoretti/Protocol/Mouse_IHC/Antibodies for mouse IHC.html〉; Haines et al., 2001; Meyer et al., 1979; Mikaelian et al., 2004; Mikaelian et al. 〈http://tumor.informatics.jax.org/mtbwi/immunohistochemistry.jsp〉; K. Rogers, M. R. Anver, D. H. Haines, 〈http://web.ncifcrf.gov/rtp/lasp/phl/immuno/〉; Shetye et al.,1966).

Use of paraffin sections provide the easiest method for daily use and especially for retrospective use. A big advantage of paraffin sections for IHC is that one can visualize morphologic changes in cells and tissues and compare them to patterns of gene expression. Many antibodies against human antigens are successfully used with paraffin sections for human IHC, but considerably lower numbers are also effective in rodents. We and others have used specific antibodies for mouse and rat IHC for many years with paraffin sections, especially for screening the effects in lymphoid tissues; they are noted in Tables 1 and 2. For rats, less protocols have been developed for use with paraffin sections but several antibodies do work for paraffin-embedded rat tissues (Shetye et al., 1966; Whiteland et al., 1995). Our antibody screens for normal cell and tissue expression and abnormal changes in these tissues are shown in our figures (Figures 1–85). An example of a complete IHC protocol of a commonly used antibody is in Table 3. Figures 1–85 show examples of commonly used antibodies for rats and mice, most with paraffin sections. Some of the rat figures are from frozen sections, but the same antibodies are also successful with paraffin sections as noted in Table 2. The specific IHC technique and chromogen used varied for each figure shown. Most figures used hematoxylin as a counterstain.

Basic IHC screens for T and B lymphocytes and cells of the mononuclear series are most important to characterize the basic responses to toxins. More specific cell types can be detected with frozen sections and sometimes with paraffin sections.

Importance of Antigen Retrieval Methods for Paraffin Sections

One of the challenges of Immunohistochemistry (IHC) is to develop methods that reverse changes produced during fixation. Antigen retrieval (AR) techniques reverse at least some of these changes. AR is particularly necessary when tissues are fixed in cross-linking fixatives, such as 10% neutral buffered formalin. Approximately 85% of antigens fixed in formalin require some type of AR to optimize the immunoreaction. A review by Romas-Vara (2005) provides much information, some of which is summarized here for use in rodent pathology. The two most common AR procedures used in IHC are enzymatic and heat-based retrieval.

Antigen Retrieval Using Enzymes

Protease-induced epitope retrieval (PIER) was introduced several years ago. Many enzymes have been used for this purpose, including trypsin, proteinase K, pronase, ficin, and pepsin. The effect of PIER depends on the concentration and type of enzyme, incubation parameters (time, temperature, and pH), and the duration of fixation. Use of a commercially available, ready-to-use solution of proteinase K that has good activity at room temperature can be used with automatic stainers. The disadvantages of PIER are the rather low number of antigens for which it is the optimal AR method, possible alteration of tissue morphology, and possible destruction of epitopes.

The following solutions are used in PIER methods: Proteinase K, Trypsin, Pepsin, Pronase or Protease 〈http://www.ihcworld.com/introduction.htm#ar〉. These methods have been reported for restoring immunoreactivity to tissue antigens with different degrees of success. However, the use of enzyme digestion methods may destroy some epitopes and tissue morphology. Therefore the optimal enzyme concentration and incubation time needs to be established.

Proteinase Kand Trypsin methods are commonly used. The concentration, incubation time and temperature probably are the most important factors to consider:

1. Concentration of enzyme is usually 0.05–0.1% depending on type of tissue and fixation.

2. Incubation time can be 5–30 minutes but 10–15 minutes is most commonly used.

3. Incubation temperature is usually at 37°C

Heat-Induced Epitope Retrieval

After deparaffinizing and rehydrating the tissue section, the slides are immersed in an aqueous solution commonly referred to as retrieval solution. Heat-induced epitope retrieval (HIER) was introduced several years ago. It is based on a concept developed by investigators who documented that the chemical reactions between proteins and formalin can be reversed, at least in part, by high temperature or strong alkaline hydrolysis. The mechanism involved in HIER is unknown, but its final effect is the reversion of conformational changes produced during fixation.

Key factors to be considered when performing pretreatment with HIER are: (1) the pH value of the retrieval solution that is dependent on the solution you are using, (2) the incubation time should be at least 10 minutes but is usually around 20 minutes, and (3) the temperature of retrieval solution which should be around 95°C. The following solutions are commonly used for HIER pretreatment: Hydrochloric Acid (pH 1.0), Formic Acid (pH 2.0), Citrate Buffer (pH 6.0), citrate-EDTA Buffer (pH 6.2), EDTA (pH 8.0), Tris-EDTA (pH 9.0), TBS (pH 9.0), Tris Buffer (pH 10).

The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagents that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. The techniques involved the application of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution (commonly referred to as the retrieval solution). This is called heat Induced epitope retrieval (HIER).

Although many different chemicals have been proposed, most retrieval solutions share a pH near 2, 6, 8, or 10. Recent systematic comparisons of several retrieval solutions showed that 0.01 M TRIS-HCL, pH 1 or 10, was slightly superior to citrate buffer of pH 6.0 and gave the best overall results 〈http://www.ihcworld.com/introduction.htm#ar〉.

Microwave oven, pressure cooker, and steamer are the most commonly used heating methods. Other tools also include the use of an autoclave or water bath. The heating length of 20 minutes appears to be the most satisfactory, and the cooling usually takes about 20 minutes. Citrate buffer of pH 6.0 is the most popularly used retrieval solution and is suitable for most of antibody applications. The TRIS-EDTA of pH9.0 and EDTA of pH8.0 are second most used retrieval solutions. Proteinase K is an effective enzyme digestion reagent for membrane antigens such as Integrins, CD31, vWF, etc.

Heating at a high temperature (100°C) for a short duration (10 minutes) gives better results than those achieve with a comparatively low temperature for a longer time. However, satisfactory results are obtained in a steamer (90–95 degrees) with a 20-minute incubation for the majority of antigens needing HIER. Several HIER solutions made of different buffers (e.g., citrate, Tris) and with various pH (3–10) levels have been used. Sometimes multiple AR methods are needed to optimize the immunodetection of antigens. A combination of Heat Mediated and Proteolytic Enzyme Method is an alternative approach to unmask antigens if other methods do not work. It is especially useful for co-localization when double or triple labeling for two or three antigens co-localization is needed.

IHC for Characterizing Immunotoxicologic Pathology: Changes in Expression of Immune System Genes

Immunotoxicity and other induced lesions (e.g., infectious diseases) can be characterized by degenerative, necrotizing and proliferative changes in thymus, lymph nodes, spleen, Peyer’s patches and other tissues. Age-related changes also occur and reactive lesions in spleen and lymph nodes have been characterized in general (Ward, 1990a, 1990b, 1993 and other papers in this issue of the journal). Protein expression at an anatomical and cellular level can be measured subjectively in the microscope or by quantitative methods. IHC changes can reflect acute, subacute and chronic changes in tissues. Acute or chronic cell loss can be seen by loss of cells expressing marker antigens in a specific tissue, especially in a specific anatomical structure of that tissue (Table 4). Modification of the normal protein expression pattern in a tissue can also show changes in tissue patterns. Changes in cell or antigen distribution can be seen. Finally, proliferative lesions or lesions in which infiltrating cells are found can be found associated with increased antigen in a tissue and even in cells. All changes may be quantified using some type of automated image analysis.

IHC findings can serve as an adjunct to histopathology and biochemical and molecular findings. The total picture of immunotoxicity can be best understood by evaluation of all research findings in a study. Conclusions as to effects of dose response, cell and organ-specific toxicity and mechanisms of toxicity can best be studied by evaluation of such findings.