ARTICLE |
Correspondence to: Ingrid A. Schrijver, Dept. of Immunology, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 TNPFicoll 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 determinanttissue 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 determinanttissue combinations. This provides additional flexibility for in situ analysis of immune reactivity. (J Histochem Cytochem 48:95103, 2000)
Key Words: acetone, immunohistochemistry, antibody-forming cells, morphology, multiple sclerosis, experimental autoimmune encephalitis, endogenous alkaline phosphatase, antigen preservation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
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 (
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) ( and IFN-
, and the novel cytokine IL-18, also known as interferon-
inducing factor (IGIF) and IL-1
(
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 (
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 (
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 TNPFicoll (a thymus-independent Type 2 antigen) or agonistic anti-CD40 antibody.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human and Animal Tissues
All mouse markers (except CD40) were examined in spleens of BALB/c mice immunized IV with 20 µg TNPFicoll, 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 (
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 (
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 ). 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, antigenenzyme conjugate (TNP-AP) or enzymesubstrate 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 TNPAP (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 TNPAP 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (
|
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 TNPFicoll-immunized mice. Anti-TNP plasma cells were present in groups of 1050 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).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.,
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 (
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 determinanttissue 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 (
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 markertissue 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.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Burnstone M (1962) Enzyme Histochemistry and Its Application in the Study of Neoplasms. New York, Academic Press
Claassen E, Gerritse K, Laman JD, Boersma WJ (1992) New immunoenzyme-cytochemical stainings for the in situ detection of epitope specificity and isotype of antibody forming B cells in experimental and natural (auto) immune responses in animals and man. J Immunol Methods 150:207-216[Medline]
Claassen E, Kors N, Van Rooijen N (1986) Influence of carriers on the development and localization of anti-2,4,6-trinitrophenyl (TNP) antibody-forming cells in the murine spleen. II. Suppressed antibody response to TNP-Ficoll after elimination of marginal zone cells. Eur J Immunol 16:492-497[Medline]
De Jong JP, Voerman JS, Leenen PJ, Van der SluijsGelling AJ, Ploemacher RE (1991) Improved fixation of frozen lympho-haemopoietic tissue sections with hexazotized pararosaniline. Histochem J 23:392-401[Medline]
Durie FH, Fava RA, Foy TM, Aruffo A, Ledbetter JA, Noelle RJ (1993) Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 261:1328-1330[Medline]
Feldmann M, Brennan FM, Maini RN (1996) Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14:397-440[Medline]
Gerritse K, Laman JD, Noelle RJ, Aruffo A, Ledbetter JA, Boersma WJ, Claassen E (1996) CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci USA 93:2499-2504
Hancock WW, Becker GJ, Atkins RC (1982) A comparison of fixatives and immunohistochemical technics for use with monoclonal antibodies to cell surface antigens. Am J Clin Pathol 78:825-831[Medline]
Hazenberg MP (1995) Intestinal flora bacteria and arthritis: why the joint? Scand J Rheumatol Suppl 101:207-211[Medline]
Hazenberg MP, Klasen IS, Kool J, Ruselervan Embden JG, Severijnen AJ (1992) Are intestinal bacteria involved in the etiology of rheumatoid arthritis? APMIS 100:1-9[Medline]
Howard LM, Miga AJ, Vanderlugt CL, Dal Canto MC, Laman JD, Noelle RJ, Miller SD (1999) Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis. J Clin Invest 103:281-290
Judd MA, Britten KJ (1982) Tissue preparation for the demonstration of surface antigen by immunoperoxidase techniques. Histochem J 14:747-753[Medline]
Kool J, De Visser H, GerritsBoeye MY, Klasen IS, Melief MJ, Van HeldenMeeuwsen CG, Van Lieshout LM, RuselerVan Embden JG, Van den Berg WB, Bahr GM (1994) Detection of intestinal flora-derived bacterial antigen complexes in splenic macrophages of rats. J Histochem Cytochem 42:1435-1441
Laman JD, Claassen E, Noelle RJ (1996) Functions of CD40 and its ligand, gp39 (CD40L). Crit Rev Immunol 16:59-108[Medline]
Laman JD, Gerritse K, Fasbender M, Boersma WJ, van Rooijen N, Claassen E (1990) Double immunocytochemical staining for in vivo detection of epitope specificity and isotype of antibody-forming cells against synthetic peptides homologous to human immunodeficiency virus-1. J Histochem Cytochem 38:457-462[Abstract]
Laman JD, Kors N, Heeney JL, Boersma WJ, Claassen E (1991) Fixation of cryosections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens. Histochemistry 96:177-183[Medline]
Laman JD, van Meurs M, Schellekens MM, de Boer M, Melchers B, Massacesi L, Lassmann H, Claassen E, 't Hart BA (1998) Expression of accessory molecules and cytokines in acute EAE in marmoset monkeys (Callithrix jacchus). J Neuroimmunol 86:30-45[Medline]
Lane P (1997) Regulation of T and B cell responses by modulating interactions between CD28/CTLA4 and their ligands, CD80 and CD86. Ann NY Acad Sci 815:392-400[Abstract]
Lucchinetti CFBW, Rodriquez M, Lassmann H (1998) Multiple sclerosis: lessons from neuropathology. Semin Neurol 18:337-349[Medline]
Massacesi L, Genain CP, LeeParritz D, Letvin NL, Canfield D, Hauser SL (1995) Active and passively induced experimental autoimmune encephalomyelitis in common marmosets: a new model for multiple sclerosis. Ann Neurol 37:519-530[Medline]
Melief MJ, Hoijer MA, Van Paassen HC, Hazenberg MP (1995) Presence of bacterial flora-derived antigen in synovial tissue macrophages and dendritic cells. Br J Rheumatol 34:1112-1116[Medline]
Morris CS, Esiri MM (1998) The expression of cytokines and their receptors in normal and mildly reactive human brain. J Neuroimmunol 92:85-97[Medline]
Okamura H, Kashiwamura S, Tsutsui H, Yoshimoto T, Nakanishi K (1998) Regulation of interferon-gamma production by IL-12 and IL-18. Curr Opin Immunol 10:259-264[Medline]
Olee T, Hashimoto S, Quach J, Lotz M (1999) IL-18 is produced by articular chondrocytes and induces proinflammatory and catabolic responses. J Immunol 162:1096-1100
Panayi GS (1993) The immunopathogenesis of rheumatoid arthritis. Br J Rheumatol 32:4-14[Medline]
Rolink A, Melchers F, Andersson J (1996) The SCID but not the RAG-2 gene product is required for S mu-S epsilon heavy chain class switching. Immunity 5:319-330[Medline]
Schoenberger SP, Toes RE, van der Voort EI, Offringa R, Melief CJ (1998) T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480-483[Medline]
Sfikakis PP, Via CS (1997) Expression of CD28, CTLA4, CD80, and CD86 molecules in patients with autoimmune rheumatic diseases: implications for immunotherapy. Clin Immunol Immunopathol 83:195-198[Medline]
't Hart BA, Bauer J, Muller HJ, Melchers B, Nicolay K, Brok H, Bontrop RE, Lassmann H, Massacesi L (1998) Histopathological characterization of magnetic resonance imaging-detectable brain white matter lesions in a primate model of multiple sclerosis: a correlative study in the experimental autoimmune encephalomyelitis model in common marmosets (Callithrix jacchus). Am J Pathol 153:649-663
van Noort JM, el Ouagmiri M, Boon J, van Sechel AC (1994) Fractionation of central nervous system myelin proteins by reversed- phase high-performance liquid chromatography. J Chromatogr 653:155-161. [B]
Whiteland JL, Shimeld C, Nicholls SM, Easty DL, Williams NA, Hill TJ (1997) Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues. J Immunol Methods 210:103-108[Medline]
Wildbaum G, Youssef S, Grabie N, Karin N (1998) Neutralizing antibodies to IFN-gamma-inducing factor prevent experimental autoimmune encephalomyelitis. J Immunol 161:6368-6374