A FIXATION METHOD FOR IMPROVED ANTIBODY PENETRATION IN ELECTRON MICROSCOPICAL IMMUNOPEROXIDASE STUDIES
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1 /78/ /$02.00/0 THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY Copyright 1978 by The Histochemical Society, Inc. Vol. 26. No. 4, pp , 1978 Printed in USA. A FIXATION METHOD FOR IMPROVED ANTIBODY PENETRATION IN ELECTRON MICROSCOPICAL IMMUNOPEROXIDASE STUDIES WOLFGANG BOHN Abteilung f#{252}rvirologie, Tropeninstitut, 2 Hamburg 4, Germany Received for publication October 12, 1977, and in revised form December 29, 1977 (MS ) A fixation method for electron microscopical immunoperoxidase staining has been developed, which (a) allows penetration of antibodies through cell membranes to intracellular antigen sites, (b) provides a reasonable cell preservation and (c) does not alter the antigenic structure in too great an extent. Penetration of the antibodies has been achieved by using sapomn as a cell membrane attacking agent. The best results could he obtained after pretreatment of cell monolayers with a mixture of 0.05% saponin, %-0.05% glutaraldehyde and 1% paraformaldehyde for 5 mm at 4#{176}C, and postfixing them with the corresponding fixative without saponin for 45 mm at 4#{176}C. One of the main obstacles in demonstrating intracellular antigen sites using immunocytochemical methods consists of the low penetration rate of antibodies through cell membranes (17). In most cases the stabilizing effects of the fixatives only allow a surface reaction. For instance, Cardiff and Lund (2) showed that they found an intracellular reaction only if the cell membrane had been destroyed. To avoid these difficulties in penetration, reactions on ultrathin frozen sections and the postembedding staining have been tried, but both methods are far from being routine (8, 18). Several attempts have been made to improve the permeability of the cell membrane for antibodies such as enzymatic digestion of the cell surface (9), freezing and thawing (10) and the application of digitonin as a cell membrane disrupting agent (3). These attempts resulted either in a heavy destruction of the cell or the penetration was too low. To continue these studies by the use of the immunoperoxidase method (12) poxvirus-infected cells seem to be most suitable, as this family of viruses produces distinct inclusions situated in the cytoplasm (11). Glutaraldehyde can be considered as the fixative of choice in electron microscopy and therefore, the author tried to develop a fixation method that (a) allows a good penetration of antibodies through cell membranes, (b) results in a satisfactory morphologic preservation and (c) does not alter the antigenic structure too This work will be part of a thesis in partial fulfillment of the requirements for the doctor s degree in biology at the University of Hamburg. much. Saponin, whose effect on cell membranes has thoroughly been examined (6, 14), has been chosen for better antibody penetration. MATERIALS AND METHODS Cell culture and virus: BHK-21 cells infected with the Shope fibroma virus (4), a member of the Leporipoxviruses, have been used as a model system. The cells were grown on coverslips in Leighton tubes. Antisera: For the indirect immunoperoxidase technique, a rabbit and anti-shope fibroma virus hyperimmune serum and a peroxidase-labeled (goat) antirabbit IgG (Miles Yeda Ltd. Rehovoth, Israel) have been used. Both sera were preabsorbed with uninfected cell monolayers. Fixation procedure: Postinfection (24 hr) the infected cells were rinsed for a short time with 0.2 M phosphate buffer; ph 7.3, 300 mos. Then they were treated with saponin (Merck 7695 Erg B 6) or with mixtures of varying concentrations of saponin and aldehyde for 5 mis at 4#{176}C, rinsed again for a short time with buffer and postflxed with the corresponding fixative without saponin. For fixation a mixture consisting of glutaraldehyde and 1% paraformaldehyde in phosphate buffer according to Robertson et al. (13) was applied. In the sapornn-aldehydmixture the concentration of saponin varied from to 0.1% and the concentration of glutaraldehyde from to 1%. Paraformaldehyde was always used at a concentration of 1%. All dilutions and further washings were done with the above mentioned buffer. Immunocytochemical staining: After fixation, the cells were rinsed for 30 min at 4#{176}C. All the following procedures were performed at room temperature. The cells were incubated with antiserum for 45 min, rinsed for 45 mis, treated with peroxidase-labeled (goat) anti-rabbit IgG for 45 mis and washed again with buffer for 45 mm. The stain reaction mixture consisted of 0.03% dia- 293
2 294 BOHN ,., I,tt - ; -. p. I1 3, 4 2 * _.J4
3 ANTIBODY PENETRATION IN IMMUNOPEROXIDASE STUDIES 295 minobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 Tris buffer (ph 7.6) (5). With this mixture the monolayers were incubated for mm. After another washing for 20 miii the cells were postflxed with 1% OsO4, dehydrated with ethanol and embedded in Epon 812. The following procedures were performed to serve as controls: (a) infected cells were reacted with peroxidase conjugate alone, (b) infected cells were reacted with unconjugated (goat) anti-rabbit IgG before the application of the peroxidase conjugate, (c) uninfected cells were reacted with immune serum and peroxidase conjugate. RESULTS Preliminary experiments using a fixative consisting of 1% paraformaldehyde and % glutaraldehyde did not allow any intracellular immunocytochemical staining, even at an incubation time of 18 hr for both sera. Therefore, saponin was chosen to enhance penetration of the antibodies through the cell membrane. The use of 0.1% saponin alone before fixation with 1% paraformaldehyde and % glutaraldehyde indeed led to an intracellular reaction (Fig. 1), but cell morphology proved to be unsatisfactory. Parts of the cell membrane were missing and the cytoplasm was destroyed to a great extent. Cell preservation was better, when saponin and aldehyde were combined (Fig. 2), i.e., membranes were preserved well and mitrochondria could be identified. Intracellular staining was still visible. In the following studies the influence of various glutaraldehyde and saponin concentrations on cell preservation and staining was investigated. Different saponin and glutaraldehyde concentrations were combined. The results are shown in Table I. Lowering the concentration of saponin from 0.1% down to 0.01% at a constant glutaraidehyde concentration of % resulted in an improved cell morphology without influencing the staining reaction. Below 0.01% saponin, only a reaction at the cell surface could be noticed (Fig. 3). Raising the concentration of glutaraldehyde at each of the different saponin concentrations also improved the preservation of ultrastructure, but beyond a certain glutaraldehyde concentration, intracellular and membrane reaction disappeared completely. The best results could be obtained with a mixture of 0.05% saponin, % glutaraldehyde and 1% paraformaldehyde (Fig. 4). In control preparations no staining was observed. DISCUSSION The results show that antibody penetration through cell membranes could be made possible by adding saponin to a fixative mixture consisting of paraformaldehyde and glutaraldehyde. There is a specific intracellular staining connected with a rather good cell preservation. Furthermore, with regard to the antigen distribution these results agree with irnmunofluorescence studies conducted with the above mentioned fixative and with acetone fixation in comparison. With regard to the efficiency of the different saponin concentrations at % glutaraldehyde the results closely agree with those received by others with erythrocyte membranes in electron microscopical studies. As proved by the appearance of intracellular staining, the first holes in the cell membrane must have appeared at 0.01% saponin, and at 0.1%, saponin destruction of the membranes became clearly visible. As shown with the freeze etching technique by Speth et al. (16) small holes on the etched surface of erythrocyte membranes became visible after treatment with 0.01% saponin. At 0.1% all membranes were fractured transversely. FIG Shope fibroma infected BHK 21-cells, 24 hr postinfection, stained for virus antigen by the indirect immunoperoxidase method after different fixation procedures. No heavy metal counterstain. FIG. 1. Cells pretreated with 0.1% saponin, fixed with 1% paraformaldehyde % glutaraldehyde. Note the specific staining of the virus particles (VP), despite heavy destruction of the cell. xl 1,000. FIG. 2. Cells pretreated with 0.1% saponin % glutaraldehyde + 1% paraformaldehyde, postfixed with % glutaraldehyde + 1% paraformaldehyde. Note the denser cytoplasm, the good preservation of the mitochondria (M) and the cell membrane. Virus particles (VP) still show specific staining. x12,700. FIG. 3. Cells pretreated with 0.001% saponin % glutaraldehyde + 1% paraformaldehyde, postfixed with % glutaraldehyde + 1 % paraformaldehyde. Note there is only a membrane reaction. Virus particles (VP) do not show any staining. x15,200. FIG. 4. Cells pretreated with 0.05% saponin % glutaraldehyde + 1% paraformaldehyde, postfixed with 0.05% glutaraldehyde + 1% paraformaldehyde. Note, that the density of the cytoplasm is increased furthermore. Mitochondria are preserved well and partially endoplasmatic reticulum (ER) can be noticed. There is specific staining only at the virus inclusion. x18,200.
4 296 BOHN TABLE Dependence of Staining Reaction on Saponin and Glutaraldehyde Concentration in Presence of 1.0% Paraforma1dehyde Glutaraldehyde % 0.025% 0.05% 0.1% 0.25% 0.5% 1.0% Saponun (%) /M /M /M 0.01 ±/M -/M -/M /M +/M ±/M #{247}/M +/M* +/W ± a, All cells showing inclusions exhibit intracellular staining; ±, the minority of cells showing inclusions exhibit intracellular staining; -, no intracellular staining; M, virus-specific staining at the membrane;, destruction of the membrane. I Freeze-etch electron microscopy of saponin treated membranes by Seeman et al. (15) revealed 4-5 nm wide pits in the outer surface of the membrane. It is difficult to explain the synergistic effect of glutaraldehyde and saponin shown in this study because the mechanism of saponin interaction with the cell membrane is not well understood (1). The results let one suppose that the stabilizing effect of glutaraldehyde, increasing with the concentration, decreases the efficiency of saponin correspondingly. The absence of any staining at high glutaraldehyde concentrations may be explained by an alteration of the antigenic structure, therefore, only low concentrations should be used. To compensate for this disadvantage in fixation a combination with paraformaldehyde is generally used. Paraformaldehyde, reacting less strongly with proteins, can be used with higher concentrations, but it does not preserve morphology as well as glutaraldehyde (7) and its use alone may lead to false positive and false negative staining (8). The fixation method described here proved to be a rapid and simple method for processing cell culture systems. Finally, it should be noted that the glutaraldehyde and saponin concentrations most suitable for this model system may be slightly different in other systems. LITERATURE CITED 1. Assa Y, Shany 5, Gestetner B, Tencer Y, Birk Y, Bondi A: Interaction of alfalfa saponins with components of the erythrocyte membrane in hemolysis. Biochim Biophys Acta 307:83, Cardiff RD, Lund JK: Distribution of dengue-2 antigens by electron immunocytochemistry. Infect Immun 3:1699, Dales 5, Gomatos PJ, Hsu KC: The uptake and development of reovirus in strain L-cells followed with labeled viral ribonucleic acid and ferritunantibody conjugates. Virology 25:193, Febvre H: The Shope fibroma virus of rabbits, Tumors Induced by Viruses, Ultrastructural Studies. Edited by AJ Dalton, F Haguenau. Academic Press, New York, 1962, p Graham RC, Kannovsky MK: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291, Graham RC, Karnovsky MJ, Schafer AW, Glass EA, Karnovsky ML: Metabolic and morphological observations on the effect of surface-active agents on leukocytes. J Cell Biol 32:629, Hayat MA: Specimen preparation, Electron Microscopy of Enzymes. Principles and Methods. Vol 1. Edited by MA Hayat. Van Nostrand Reinhold Company, New York 1973, p Kuhlmann WD: Ultrastructural Immunoperoxidase Cytochemistry. Progress in Histochemistry and Cytochemistry Vol 10, No. 1. Gustav Fischer Verlag, Stuttgart, New York, Kuhlmann WD, Avrameas 5, Ternynck T: A comparative study for the ultrastructural localization of intracellular immunoglobulins using peroxidase conjugates. Immunol Meth 5:33, Leduc EH, Scott GB, Avrameas 5: Ultrastructural localization of intracellular immune globulins in plasma cells and lymphoblasts by enzyme-labeled antibodies. J Histochem Cytochem 17:211, Morgan C, Ellison SA, Rose HM, Moore DH: Structure and development of viruses observed in the electron microscope II. Vaccinia and fowlpox viruses. J Exp Med 100:301, Nakane PK, Pierce GBJ: Enzyme-labeled antibodies for the light and electron microscopic localization of tissue antigens. J Cell Biol 33:307, Robertson JD, Bodenheimer TS, Stage DE: The ultrastructure of Mauthner cell synapses and nodes in goldfish brains. J Cell Biol 19:159, Seeman P: Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin. J Cell Biol 32:55, 1967
5 ANTIBODY PENETRATION IN IMMUNOPEROXIDASE STUDIES Seeman P, Cheng D, Iles GH: Structure of membrane holes in osmotic and saponin hemolysis. J Cell Biol 56:519, Speth V, Wallach DFH, Weidekamm E, Kn#{252}fermann H: Micromorphologic consequences following perturbation of erythrocyte membranes by trypsin, phospholipase A, Lysolecithin, sodium dodecylsulfate and saponin. Biochim Biophys Acta 255:386, Sternberger LA: Immunocytochemistry. Prentice- Hall, Inc, Englewood Cliffs, N.J., Vogt A, Takamiya H, Kim WA: Some problems involved in postembedding staining, Immunoenzymatic Techniques. Edited by S Avrameas, S Bignon, P Druet, G Feldmann. North-Holland Publ. Co., Amsterdam, 1976, p
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