ARTICLE

Pararosaniline Fixation for Detection of Co-stimulatory Molecules, Cytokines, and Specific Antibody

Correspondence to: Ingrid A. Schrijver, Dept. of Immunology, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands.

	Summary

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Integral immunohistochemical analysis of immune responses in frozen sections requires that, in addition to constitutively expressed membrane CD markers, less stable determinants can be reliably visualized. Therefore, we compared the commonly used acetone fixation method with pararosaniline fixation for six determinant categories. These categories included selected constitutively expressed markers, inducible co-stimulatory molecules, pro- and anti-inflammatory cytokines (including the novel cytokine IL-18, also known as IGIF and IL-1), antigen-specific antibody in plasma cells, bacterial peptidoglycan, and lysosomal acid phosphatase activity. Human spleen and mouse spleen activated by agonistic anti-CD40 antibody or TNP–Ficoll immunization were analyzed in parallel with brain tissue from multiple sclerosis (MS) patients and marmoset monkeys with experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Fixation with pararosaniline resulted in better morphology of all tissues and inhibited endogenous alkaline phosphatase activity in brain tissue. Most determinants could be reliably detected. Staining sensitivity and intensity were markedly increased for selected determinant–tissue combinations, e.g., for IL-4 in human spleen and CD40 in human and mouse spleen. These data show that pararosaniline is a useful alternative to acetone, resulting in superior morphology and specific staining for selected determinant–tissue combinations. This provides additional flexibility for in situ analysis of immune reactivity. (J Histochem Cytochem 48:95–103, 2000)

Key Words: acetone, immunohistochemistry, antibody-forming cells, morphology, multiple sclerosis, experimental autoimmune encephalitis, endogenous alkaline phosphatase, antigen preservation

	Introduction

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CHRONIC INFLAMMATORY DISEASES such as rheumatoid arthritis (RA) and multiple sclerosis (MS) are generally believed to result from the activity of autoreactive CD4-positive T-cells. The formation and activity of lesions caused by infiltrating mononuclear cells are complex processes that are incompletely understood, and this also holds true for the cellular effector mechanisms that might be responsible for the tissue damage that leads to temporary and cumulative clinical symptoms. Such effector functions include local (auto)-antigen presentation (auto)-antibody production, secretion of pro- and anti-inflammatory cytokines, and activity of enzymes involved in proteolysis and nitric oxide (NO) production (Panayi 1993 ; Lucchinetti et al. 1998 )

It is therefore crucial to analyze inflammatory lesions functionally in situ by conventional and novel immunohistochemical methods to provide an integral view of ongoing cellular activities. Relevant parameters are the localization of antigen possibly involved in inflammation [e.g., bacterial peptidoglycan (PG) in RA]. PG may be involved in the pathogenesis of chronic inflammation, particularly in RA (Hazenberg et al. 1992 ; Hazenberg 1995 ). With a monoclonal antibody (MAb) against PG (2E9), antigen-presenting cells containing PG can be detected in synovial tissues (Kool et al. 1994 ; Melief et al. 1995 ). Furthermore, the integrity of antigen binding sites of antibodies present in the cytoplasm of plasma cells can be evaluated (Laman et al. 1990 , Laman et al. 1991 ; Claassen et al. 1992 ). This was studied by detection of antibody-forming cells specific for 2,4,6-trinitrophenyl (TNP) in mice immunized with TNP–Ficoll (Claassen et al. 1986 ).

The identification of mononuclear cell subsets [e.g. T-, B-, and antigen-presenting cells (APCs), granulocytes] by markers such as IgM, IgG, CD14, and HLA-DR or enzyme activity (lysosomal acid phosphatase in macrophages) can be used to examine the presence of B-cells and antigen-presenting cells that can contribute to pathogenesis of RA and MS by producing antibodies and cytokines or enzymes. Functional activation profiles can be identified by strongly regulated co-stimulatory molecules such as CD40, CD40L, CD80 (B7-1), and CD86 (B7-2) (Laman et al. 1996 ; Lane 1997 ), which have been claimed to play an important role in the inflammatory process of both MS (Gerritse et al. 1996 ; Howard et al. 1999 ) and RA (Durie et al. 1993 ; Sfikakis and Via 1997 ). Other important activation markers in immunological responses are intracytoplasmic soluble cytokines (Feldmann et al. 1996 ; Morris and Esiri 1998 ) such as the anti-inflammatory cytokines IL-4 and IL-10, the proinflammatory cytokines TNF- and IFN-, and the novel cytokine IL-18, also known as interferon- inducing factor (IGIF) and IL-1 (Okamura et al. 1998 ; Wildbaum et al. 1998 ; Olee et al. 1999 ).

To identify all these relevant parameters involved in inflammation by immunohistochemistry, adequate fixation of frozen sections is an essential requirement. The main function of fixation is to preserve morphology of the tissue, which is often best accomplished by strong fixation. The disadvantage of strong fixation is that the structure and integrity of the parameters are changed, resulting in poor quality of the staining pattern. The optimal fixation condition therefore consists of a compromise between these contradictory requirements and may differ depending on the nature of the antigen (De Jong et al. 1991 ).

Pararosaniline has previously been shown to have good fixation properties for frozen tissue sections without affecting antigenicity. This was concluded from staining for constitutively expressed markers in mouse lymphohemopoietic organs in which acetone fixation was compared with pararosaniline fixation (De Jong et al. 1991 ).

The aim of this study was to determine whether pararosaniline has advantages over acetone fixation for detection of less stable determinants such as bacterial antigen, intracellular cytokines, co-stimulatory molecules, and specific antibody located in plasma cells. Expression of the markers was examined in different types of organ tissues (spleen, brain) from three species (human, mouse, and monkey). Tissues were activated either by chronic inflammation or by in vivo administration of TNP–Ficoll (a thymus-independent Type 2 antigen) or agonistic anti-CD40 antibody.

	Materials and Methods

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Human and Animal Tissues
All mouse markers (except CD40) were examined in spleens of BALB/c mice immunized IV with 20 µg TNP–Ficoll, a polysaccharide thymus-independent Type 2 antigen which evokes IgM and IgG antibody-forming cells, expression of CD40L (CD154), and several cytokines 4 days after injection (Claassen et al. 1986 ). CD40 detection was performed on C57Bl/6 spleen in which CD40 expression was upregulated by IV injection of 100 µg agonistic anti-CD40 antibody (FGK-45) (Rolink et al. 1996 ; Schoenberger et al. 1998 ). At 96 hr after injection, the mice were sacrificed and spleen tissue was removed.

To study expression of co-stimulatory molecules and cytokines in tissue affected by chronic autoimmune inflammation, brain tissue from marmoset monkey EAE (Callithrix jacchus) was used. The monkeys were bred at the Biomedical Primate Research Center (BPRC) and used under conditions approved by Dutch laws on animal experimentation. EAE was induced essentially as described previously (Massacesi et al. 1995 ; Laman et al. 1998 ; 't Hart et al. 1998 ). Per animal, 20 mg of human myelin (van Noort et al. 1994 ) in PBS was emulsified with an equal volume of complete Freund's adjuvant containing 3 mg/ml of Mycobacterium tuberculosis (H37 RA strain). Intracutaneous injections of 150 µl were divided over four sites on the dorsal axillary and inguinal regions. Animals were sacrificed during active disease periods and brain tissue was obtained.

Expression of human markers was determined in human spleen without evidence of infection or inflammation. Two human spleens were removed after damage to the spleen occurred during surgery for a stomach carcinoma.

To study expression of co-stimulatory molecules and cytokines in human tissue affected by chronic autoimmune inflammation, brain tissue of MS patients was used for determination of all cytokines and co-stimulatory molecules expected to be expressed in lesions. Two human MS brain tissue specimens were obtained at autopsy with short postmortem intervals from the Netherlands Brain Bank in Amsterdam, The Netherlands (coordinator Dr. R. Ravid).

Tissue Processing
All tissues were snap-frozen in liquid nitrogen in aluminum containers, which were stored at -80C. Six-µm frozen sections were cut on a cryostat and thaw-mounted on slides precoated with 0.1% gelatin (Merck; Darmstadt, Germany) and 0.01% chromium potassium sulfate (Merck). The sections were kept overnight in a box with a humidified atmosphere, after which they were air-dried at room temperature (RT) for 1 hr and stored in a box with silica until use.

Fixatives
Acetone (purity <99.5%) (Fluka Chemie; Buchs, Switzerland) supplemented with 0.5% H₂O₂ (from a 30% stock solution) was used at RT. Pararosaniline was prepared as described by Burnstone 1962 with minor modifications (De Jong et al. 1991 ). Four percent (w/v) pararosaniline (Sigma Chemical; St Louis, MO) was dissolved in 2 M HCl by heating at 70C. After the pararosaniline was dissolved, the solution was filtered and 1 ml of this solution was mixed with 1 ml 4% (w/v) NaNO₂ in distilled water. After precisely 1 min this reaction mixture was dissolved in 200 ml MilliQ water (Millipore, Bedford, MA; PF+ system, resistance >18 M). This solution can be used for at least up to 1 month when stored at 4C. Pararosaniline and solutions containing this chemical should be handled with caution in view of their potential carcinogenicity.

(Immuno)histochemistry
Sections were fixed according to the different protocols (Table 1). The slides were next washed with PBS and then with PBS/0.05% (v/v) Tween-20 (Fluka) and incubated with the first reagent [antibody, antigen–enzyme conjugate (TNP-AP) or enzyme–substrate solution] in PBS/1% BSA/0.05%Tween (HPLC grade, fraction V; Sigma) (Table 2) overnight at 4C (or 30 min at 37C for the acid phosphatase substrate). After washing the slides twice with PBS/0.05% Tween, the sections were incubated with the appropriate secondary reagent (PBS/1%BSA/0.05% Tween/1% normal human serum) for 30 min at RT (Table 2). For some stainings, the slides were then washed twice with PBS/Tween and incubated for 30 min with the tertiary reagent (Table 2). After the slides were rinsed twice in PBS, they were washed in 0.05 M NaAc buffer, pH 5.0, for 5 min[except for the TNP–AP (alkaline phosphatase) staining]. Horseradish peroxidase activity (HRP) was revealed by incubating the slides for 15 min in substrate containing 1.5 ml of 3-amino-9-ethylcarbazole (AEC; Sigma) dissolved in N,N-dimethylformamide (DMF; Sigma) (17 mg/ml) and 30 µl H₂O₂ 30% in 60 ml of NaAc buffer, pH 5.0, followed by a washing step in PBS. The use of AEC results in a bright red precipitate. The histochemical revelation of TNP–AP staining was performed with Fast Blue Base (FBB) substrate after rinsing the slides in 0.1 M Tris-HCl, pH 8.5. FBB substrate was prepared as follows. Sixteen mg FBB (Sigma) was added to 400 µl 2M HCl and 400 µl 4% NaNO₂ and was mixed for 1 min before adding to 65 ml of 0.1 M Tris-HCl, pH 8.5. Then 8 mg naphthol-AS-MX phosphate (Sigma) was dissolved in 400 µl of DMF and added to the solution. Finally, 100 µl of 1 M levamisole (Sigma) was added to the solution to inhibit endogenous AP activity. The slides were incubated in the substrate for 45 min. The slides were washed in NaAc buffer, pH 5.0, or in 0.1 M TRIS-HCl, pH 8.5. All slides were counterstained with hematoxylin (Mayer's) and washed for 10 min in tapwater, after which they were embedded in Kaiser's glycerin/gelatin (Merck).

Evaluation and Quantitation of (Immuno)histochemistry
Staining on two sections of each tissue was performed for each marker. All sections were evaluated for the following parameters; tissue morphology, sensitivity of the procedure as defined by number of positive cells, staining intensity per cell, and background endogenous enzyme activity (HRP and AP). The evaluation was performed by light microscopy by two independent observers blinded to the fixation procedure.

	Results

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Morphology
To assess the preservation of tissue and cell morphology after the different fixation procedures, the tissue sections were stained using the entire staining procedure but with omission of the primary reagent. As described previously (De Jong et al. 1991 ), after acetone fixation the morphology was suboptimal for mouse and human spleen. The distinction between red and white pulp was not very clear and the distinction between individual cells was not optimal, especially in the white pulp (Figure 1A and Figure 1B). The morphology of monkey EAE brain was reasonable after acetone fixation except for the mononuclear cell infiltrates, in which the individual cells were not clearly discernible. Morphology of MS brain was good in unaffected parts but suboptimal in demyelinating parts of the tissue.

View larger version (124K): [in this window] [in a new window]   Figure 1. Morphology and endogenous AP activity after acetone and pararosaniline fixation. Morphology of human spleen tissue strongly improved after pararosaniline (B) fixation compared to acetone fixation (A). After pararosaniline fixation, the distinction between red (RP) and white pulp (WP) is much clearer and hematoxylin counterstaining is stronger. Endogenous AP activity of endothelial cells of EAE brain could be observed after acetone (C). After pararosaniline fixation, endogenous AP activity was totally inhibited (D). Bar = 20 µm.

In all tissues (except for MS brain), the morphology improved after fixation with pararosaniline. For MS brain tissue there was no difference between the two fixatives. After pararosaniline fixation, both densely populated areas, such as the splenic white pulp, and less densely populated areas (red pulp) of the spleen and infiltrates of EAE brain showed better morphology. At low-power magnification, the distinction between spleen white and red pulp could be made much more easily after pararosaniline fixation compared to acetone. In addition, the distinction between individual cells and determination of cell type on the basis of morphology was much easier after pararosaniline fixation compared to acetone fixation. After pararosaniline fixation, the counterstaining of the nuclei with hematoxylin was more intense compared to acetone fixation.

Endogenous Enzymatic Activity
To determine whether endogenous peroxidase or AP activity could be inhibited by pararosaniline or acetone fixation, all tissues were stained with omission of the primary antibody and the histochemical revelation of HRP and AP activity was performed with AEC substrate and Fast Blue Base substrate, respectively. Endogenous peroxidase activity was routinely inhibited by incubation with 0.05% H₂O₂ and endogenous AP activity with levamisole added to the substrate.

In none of the tissues could endogenous peroxidase staining be observed after acetone or pararosaniline fixation. In human and mouse spleen, endogenous AP activity was completely blocked after both fixation procedures. In contrast, EAE and MS brain tissue showed a difference between the fixation procedures. After acetone fixation, endogenous AP activity was present in the endothelial cells of the blood vessels in MS brain and monkey EAE brain. After pararosaniline fixation this endogenous AP activity was completely abolished (Figure 1C and Figure 1D).

Preservation of Determinants
To examine the effect of acetone and pararosaniline on antigen preservation, detection of a broad range of different determinants on various tissues was analyzed. The antigen preservation after the fixation method was analyzed by two parameters. First, the number of cells positive for the antigen was counted and, second, the staining intensity on a per cell basis was determined. The semiquantitative results are shown in Table 3. The staining properties were dependent on the combination of marker and tissue used. Many of the markers showed the same staining pattern after both fixation procedures. However, some markers showed considerably different staining patterns for the two fixation procedures. For example, pararosaniline is preferable for detection of CD40 in all tissues. For other markers, the difference between the fixatives is restricted by the tissue used. For example, for IL-10 detection pararosaniline fixation is preferable for marmoset brain but not for human MS brain. In addition, in the six categories of markers described above there is no general rule as to which fixative is preferable. For example, higher numbers of IL-4 producing cells were detected with pararosaniline in human spleen but no IL-18 could be detected. The general impression is that, for human and mouse spleen tissue, pararosaniline is the better fixative. The staining of the cells is more discrete compared to acetone, with which the staining is more diffuse with some color precipitate outside the cell. Therefore, it is sometimes difficult to determine which individual cell is positive for the determinant under investigation, which may lead to an overestimation of the number of positive cells after acetone fixation.

Especially with markers present on a high number of cells, such as CD14 and HLA-DR, the staining intensity of the cells was higher after pararosaniline fixation. A disadvantage of pararosaniline fixation is that no positive cells could be observed for IFN- and IL-18 in human spleen and for CD86 in MS brain.

Figure 2 shows some selected stainings representing the different categories of determinants used in the study. Lysosomal acid phosphatase activity can be detected histochemically in all macrophages present in the EAE brain tissue. Pararosaniline fixation resulted in similar staining intensity compared to acetone fixation (Figure 2A and Figure 2B). IL-4 is expressed in the red pulp of human spleen. The staining pattern is more discrete and stronger after pararosaniline compared to acetone fixation (Figure 2C and Figure 2D). CD40 expression in mouse spleen injected with the agonistic anti-CD40 antibody is weak on B-cells in the white pulp and strong on some macrophages in the red pulp. After pararosaniline, the staining of the B-cells is more intense compared to acetone fixation. (Figure 2E and Figure 2F). Plasma cells containing intracellular antibody specific for TNP were detected in TNP–Ficoll-immunized mice. Anti-TNP plasma cells were present in groups of 10–50 cells located in the outer pals and terminal arterioles of the spleen. Staining patterns did not differ between the two fixation procedures, indicating that pararosaniline does not affect the integrity of antigen binding sites, but morphology of the tissue strongly improved after pararosaniline fixation (Figure 2G and Figure 2H).

View larger version (136K): [in this window] [in a new window]   Figure 2. Antigen preservation of different markers after acetone and pararosaniline fixation. Acetone (left column)- and pararosaniline (right column)-fixed sections of noninfiltrated part of EAE brain (A,B), human spleen (C,D), and mouse spleen (E–H). Macrophages containing lysosomal acid phosphatase were located throughout the sections (A,B). No difference of staining pattern was found between the two fixatives. IL-4-producing cells (C,D) were mostly located in the red pulp of the spleen. After pararosaniline fixation, staining intensity per cell was improved compared to acetone, in which the staining was more diffuse. CD40 was expressed on B-cells and some macrophages (E,F). After pararosaniline fixation, expression of CD40 on B-cells was much stronger compared to acetone fixation. Plasma cells specific for TNP were analyzed in TNP–Ficoll-injected mice (G,H). Groups of 10–50 cells were detected in the outer pals and terminal arterioles of the spleen. Pararosaniline fixation did not affect integrity of antigen binding sites of antibodies, but morphology was strongly improved. Bars = 20 µm.

	Discussion

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The present data clearly demonstrate that pararosaniline is a permissive and mild fixative for frozen sections, which can result in improved immunohistochemical staining compared to acetone, depending on the combination of the marker and the tissue under study.

The purpose of this study was to identify improved fixation conditions for the study of immune responses in situ. Integral immunohistochemical analysis of immune responses requires that, in addition to constitutively expressed membrane CD markers, strongly regulated determinants such as bacterial antigen, intracellular cytokines, co-stimulatory molecules, enzyme activity, and antigen-specific plasma cells can be visualized reliably. Although some studies have reported that unstable determinants such as cytokines are detected reliably in paraffin-embedded tissue (e.g., Whiteland et al. 1997 ), most studies of strongly regulated determinants employ frozen sections because efficient detection of these markers remains difficult in paraffin-embedded tissues. A major disadvantage of the use of frozen tissues is the frequently poor morphology of cryosections. Morphology of the tissues is strongly dependent on the fixative used. Good morphology is achieved with paraformaldehyde, formalin, and glutaraldehyde, which are able to crosslink proteins. A major disadvantage of these fixatives is the alteration of tertiary structure by crosslinkage, resulting in alteration of antigenic structures of the tissues (Hancock et al. 1982 ). In contrast, acetone, which precipitates proteins, is a reliable fixative for detection of different markers but morphology is often poor (Judd and Britten 1982 ).

To improve morphology while maintaining staining intensity, we compared the most commonly used mild acetone fixation with pararosaniline. Pararosaniline is a hexazonium salt that contains three reactive diazonium groups and therefore, in theory, must be able to crosslink proteins, which was shown previously to yield a better morphology of mouse tissue compared to acetone without affecting immunogenicity (De Jong et al. 1991 ). In the present study, detection of six categories of determinants was analyzed for different tissues. Human and mouse spleens were used because the spleen is an important secondary lymphoid organ in which a wide diversity of immune responses takes place. Therefore, most of the markers examined are expressed in these organs. We also evaluated expression of these determinant categories in inflamed tissues of MS patients and EAE-affected marmoset monkeys because it is expected to be chronically increased and because good morphology is very important in these activated tissues.

Antigen preservation was analyzed by examination of staining intensity per cell and number of positive cells. In general, the antigen preservation after pararosaniline fixation did not differ from that of acetone fixation. Staining of antigen-specific antibodies and acid phosphatase could be detected reliably in all tissues, implying that these determinants are well preserved. The preservation of strongly regulated molecules such as co-stimulatory molecules and cytokines differed between the two fixation procedures. The restriction of pararosaniline fixation is that, for a few determinant–tissue combinations, the number of cells positive for the determinant is lower compared to acetone fixation. In these cases, acetone fixation would be preferable. In all other cases, fixation with pararosaniline is preferable over acetone fixation for four reasons. First, morphology is strongly improved after pararosaniline fixation, which results in a better judgment of the location of positive cells. Second, endogenous AP activity could be completely inhibited after pararosaniline fixation. This is especially important when double stainings are performed with revelation of both peroxidase and AP enzymatic activity (Laman et al. 1990 ). Third, in some cases staining intensity after pararosaniline fixation improved, leading to a more discrete staining pattern compared to acetone fixation, in which color precipitate is sometimes seen outside the cell. Especially when high numbers of cells are positive for the determinant, this will lead to a better distinction between positive cells. Fourth, hematoxylin counterstaining of the tissue was much stronger compared to acetone fixation, leading to an improved distinction between adjacent cells.

In conclusion, pararosaniline fixation gives a better morphology than acetone fixation and inhibits endogenous AP activity in brain tissue. Antigen preservation in general does not differ between the two fixatives for a wide range of determinants. When staining protocols are established for new and existing reagents, pararosaniline is an easy and cost-effective alternative fixation for frozen sections. For studying a new marker–tissue combination, it is recommended to analyze acetone and pararosaniline fixation routinely side by side to determine the optimal fixation procedure.

	Acknowledgments

Supported by the EC Biomed-2 grant BMT4-CT-97-2131 and NWO-NDRF grant 014-80-007.

We thank Dr R. Toes for providing the anti-CD40-injected C57Bl/6 mouse spleen material, Dr B. 't Hart (BPRC; Rijswijk, The Netherlands) for providing the marmoset EAE material, and the Netherlands Brain Bank (coordinator Dr. R. Ravid) for providing the MS brain tissues. We thank all suppliers of monoclonal antibodies for their kind gifts (Drs P. van der Meide, P. Leenen, M de Boer, H.F.J. Savelkoul). Furthermore we would like to thank Dr P. Leenen and Dr B. 't Hart for critical reading of this manuscript, and T. van Os for the photomicrographs.

Received for publication June 10, 1999; accepted August 31, 1999.

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