Proc. Natl. Acad. Sci. USA Vol. 75, No. 4, pp. 1820-1824, April 1978 Cell Biology
Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy (tubulin antibody/NaBH4/fixation procedures/actin/immunocytochemistry)
KLAUS WEBER, PETER C. RATHKE, AND MARY OSBORN Max Planck Institute for Biophysical Chemistry, D-3400 Goettingen, Federal Republic of Germany
Communicated by Manfred Eigen, January 10, 1978
ABSTRACT Electron microscopy and indirect immunofluorescence microscopy using monospecific tubulin antibodies were performed in parallel on glutaraldehyde-fixed tissue culture cells without osmium fixation. In order to reduce the excess aldehyde groups of the strongly crosslinked cellular matrix, which normally interfere with subsequent immunofluorescence microscopy, a mild NaBH4 treatment was introduced during or after the dehydration steps. Cells processed through the NaBH4 step show, in transmission electron microscopy, normal cytoplasmic microtubules ap roximately 250 A in diameter. When such cells are subjected to indirect immunofluorescence microscopy using monospecific tubulin antibody they reveal a complex system of unbroken, fine, fluorescent fibers traversing the cytoplasm between the perinuclear space and the plasma membrane. Thin sections of cells processed through the indirect immunofluorescence procedure show antibody-ecorated microtubules with a diameter of approximately 600 A. This decoration is not obtained when nonimmune IgGs are used instead of monospecific antitubulin IgGs. Thus, a direct comparison of cytoplasmic microtubules in glutaraldehyde-fixed cells by both electron microscopy and immunofluorescence microscopy can be obtained. Use of specific antibodies against actin and tubulin allows the distribution of microfilament bundles (1) and the organization of cytoplasmic microtubules (2) in tissue culture cells to be demonstrated by indirect immunofluorescence microscopy. In addition, the organization of tonofilaments has been demonstrated in one cell line by using an autoimmune serum (3). The advantages of the procedure include the direct overview provided over the whole cell and the opportunity to study numerous cells simultaneously. Documentation of cytoplasmic microtubules in tissue culture cells by immunofluorescence microscopy (2-10) has indicated the following features. (i) Microtubules are present in large numbers during interphase (2, 4). (ii) Microtubules can be followed for very long distances. They traverse the cytoplasm from the perinuclear space toward the plasma membrane and can also run for long distances underneath the plasma membrane (2, 4-10). (iii) Many of these cortical microtubules seem to originate in the centrosphere acting as an organization center, and after depolymerization they appear to regrow in a unidirectional manner toward the plasma membrane (5-7). (iv) At the onset of mitosis the complex pattern of cytoplasmic microtubules is reorganized to form the microtubules of the mitotic spindle. At late telophase, or in early G1, cytoplasmic microtubules reappear in the daughter cells originating from the centrosphere (4, 8-10). Although these results confirm and extend previous data on
microtubules obtained by conventional transmission electron microscopy on small cell samples (e.g., see refs. 11 and 12) and although the use of the immunoperoxidase technique has shown that tubulin antibodies decorate cytoplasmic microtubules as ascertained by low-power electron microscopy (13), it is desirable to examine cells processed in the same manner by both immunofluorescence and electron microscopy. This is particularly necessary because the fixation procedures traditionally used in the two techniques have been different. Indirect immunofluorescence microscopy has often been criticized because of the use of formaldehyde rather than glutaraldehyde for fixation (e.g., refs. 14 and 15). Extensive fixation with glutaraldehyde (16) is considered a requirement for microtubular preservation on the ultrastructural level, and the mild formaldehyde fixation usually performed in immunofluorescence studies (1, 2) is assumed to be accompanied by a disintegration of the delicate microtubular structure either during fixation or during dehydration (14, 15). Extensive fixation with glutaraldehyde, however, has been avoided in immunofluorescence microscopy because the background staining experienced in such studies has been severe (e.g., ref. 17). A common fixation procedure satisfying both the needs of electron microscopy and the approach used in immunofluorescence microscopy has not been obtained. Here we show that glutaraldehyde-fixed cells treated with NaBH4 and then processed through the indirect immunofluorescent procedure can be viewed in either the fluorescence or the electron microscope. Immunofluorescence microscopy shows a striking display of microtubules similar to that found after formaldehyde fixation (2-10). Electron microscopy shows microtubules specifically decorated with antibody. MATERIALS AND METHODS Cells and Antibodies. Mouse 3T3 cells were grown on round 12-mm glass coverslips (5). Antitubulin antibody-was raised in rabbits against homogeneous 6S porcine cerebrum tubulin free of microtubule-associated proteins and was made monospecific (18). The arguments for the tubulin specificity of such antibodies have been summarized (10). In addition, our antibodies can be used to measure tubulin quantitatively in a radioimmunoassay (unpublished data). The fluorescein-labeled goat antibody against rabbit IgG was from Miles Yeda, Israel, and was used after 1:10 dilution into phosphate buffered saline (Pi/NaCl). The rabbit antiactin antibody has been described
(1, 19).
Preparation of Samples. Procedure 1. (a) Fix in 2.5% glutaraldehyde (Serva, Heidelberg, F.R.G.; no. 23114) in 0.1 M Na
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: Pi/NaCl, phosphate-buffered saline.
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Cell Biology: Weber et al. cacodylate/10 mM KCI/5 mM MgCl2, pH 7.2 for 1Q0.tiwiat room temperature, followed by 10 min on ice. (b) Rinse four times, 7 min each, with ice-cold 0.1 M Na cacodylate, pH 7.2. (c) Serially dehydrate through ice-cold 50%, 70%, 80%, 90%, and 96% ethanol for 15 min each. (d) Treat with NaBH4 (Merck Co., F.R.G.), 0.5 mg/ml in 96% ethanol, three times for 6 min each at 40. (Under these conditions, NaBH4, dissolves slowly; solutions were made up 3-4 min prior to the addition of the coverslips.) Wash two times for 5 min each with 96% ethanol at 46. (e) Serially rehydrate through ethanol at 50% (40), 20%, and 10% at room temperature and equilibrate with Pi/NaCl at room temperature. (f) Add antitubulin antibody (0.05 mg/ml in Pi/NaCl) and incubate 45 min at 370; wash well with Pi/ NaCl. (g) Add fluorescein-labeled goat antirabbit antibody and incubate 45 min at 370; wash well with Pi/NaCl. (h) For immunofluorescence microscopy, coverslips were mounted in Elvanol and examined with a Zeiss fluorescence microscope equipped with epifluorescent illumination (for details, see ref. 20). (i) For electron microscopic analysis, steps a, b, and c are repeated. (j) Dehydrate the cells in water-free ethanol followed by water-free propylene oxide and embed as monolayers in Epon 812. Ultrathin sections were cut parallel to the original substratum and stained with uranyl acetate and lead citrate (for details, see ref. 21) and examined with a Philips 301 electron microscope. Procedure 1 was designed to stay as close as possible to normal electron microscopy procedures. The conditions for the NaBH4 step have not yet been optimized. Preliminary experiments suggest that the NaBH4 treatment can also be done either in step c in'50% ethanol, in which case the further serial dehydration in this step can be omitted, or in Pi/NaCl after step e using steps b and c from procedure 2. Procedure 2. (a) Fix in 1% glutaraldehyde in Pi/NaCl for 15 min at room temperature; treat with methanol at -10° for 15 min. (b) Treat with NaBH4, 0.5 mg/ml in Pi/NaCl, three times for 4 min each at room temperature. (c) Wash with Pi/NaCl two times for 3 min each at room temperature. (d) Use steps f and g of procedure 1. For fluorescence microscopy, use step h above; for electron microscopy, use step i above. RESULTS AND DISCUSSION Fixation procedure 1 allows a parallel study of cytoplasmic microtubules in tissue culture cells by both immunofluorescence microscopy and electron microscopy. Furthermore, the final product used in fluorescence microscopy can also be characterized by transmission electron microscopy. The need to accommodate strong glutaraldehyde fixation in the procedure and to avoid loss of antigenicity by too-harsh chemical treatments necessitated changes in the normal electron microscopic fixation of cells (e.g., ref. 21) as well as in the normal immunofluorescence microscopy (1, 2, 20). We have tried to keep these changes to a minimum. The following points of the procedure should be discussed. 1. We have omitted postfixation with osmium tetroxide because this treatment severely hampers subsequent decoration of microtubules in immunofluorescence microscopy. Athough omission of this step decreases the preservation of membraneous structures, microtubules are still well preserved in thin sections. 2. The fixation step uses a concentration of glutaraldehyde of 2.5% and an exposure time of 20 min as prescribed in electron microscopy procedures, rather than the formaldehyde fixation previously used in immunofluorescence microscopy (2-10, 20). Dehydration was performed as in normal electron microscopy studies.
Proc. Natl. Acad. Sci. USA 75 (1978)
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3.- To be able to use glutaraldehyde fixation in indirect immunofluorescence microscopy, we had to overcome the strong nonspecific background staining, described by others (e.g., ref. 17) and experienced also by us. This background is also found when nonimmune sera are used. Here we were helped by two observations with procedure 2 omitting steps b and c: (i) glutaraldehyde-fixed cells, as well as such cells after treatment with the nonfluorescent antibody, did not show autofluorescence; (if) when tubulin antibody followed by the second fluorescent antibody was used, decoration of cytoplasmic microtubules against a very high background was always observed. The nonspecific background staining was increased when the protein concentration of either the first or the second antibody was increased, regardless if the first antibody was from an immune serum or a nonimmune serum. These results suggested that, even after extensive washing, the glutaraldehyde-fixed cell matrix could contain excess covalently bound aldehyde groups which, by binding either the first or second antibody, are responsible for the unspecific background staining seen. This assumption, which is independent of whether glutaraldehyde fixation of proteins is performed via the normal dialdehyde or via a complex polymeric product obtained by aldol condensation (for a recent review, see ref. 22), was tested by introducing a gentle aldehyde reduction step. Experiments with procedure 2 showed that treatment of the glutaraldehyde-fixed cell matrix after the methanol step with the aldehyde reducing agent NaBH4 in buffer decreased the background staining dramatically. Cytoplasmic microtubules could readily be visualized in indirect immunofluorescence microscopy against a relatively dark background (Fig. la). Electron microscopic analysis of 3T3 cells subjected to NaBH4 treatment (Fig. 2a) showed normal microfilaments, intermediate filaments, and ribosomes. Because of the lack of osmium fixation, cellular membraneous structures exhibited a slightly decreased, and in some cases (e.g., mitochondria) an apparently reversed, contrast. Microtubules were relatively well preserved and stained, although their surfaces seemed rougher than in cells subjected to osmium fixation. The diameter of the microtubules was 250 A. 4. In order to assess the influence of rehydration and indirect antibody decoration on glutaraldehyde-fixed cells subjected to NaBH4 treatment, we studied the effect of rabbit nonimmune IgGs (100-200 ;zg/ml in Pi/NaCl) followed by fluorescent goat antirabbit IgGs (1 mg/ml in Pi/NaCl) on the preservation of cytoplasmic microtubules. Electron microscopic analysis demonstrated no structural damage to the microtubules as well as preservation of their diameter at approximately 250 A (Fig. 2b). In immunofluorescence microscopy, some residual staining, particularly of the nucleus, was observed (not shown). 5. Substitution of monospecific rabbit antitubulin IgGs (0.05 mg/ml) for nonimmune rabbit IgGs (see above) followed by treatment with fluorescent goat antirabbit IgGs gave a totally different impression when ultrathin sections were examined in the electron microscope. Although a normal display of microfilament bundles, intermediate filaments, and ribosomes was observed, all microtubules were heavily stained (Fig. 2 c and d) and had diameters of approximately 600 A rather than 250 A (see above). Because IgGs have a maximum length of approximately 100 A, a microtubule decorated around its circumference first by antitubulin antibody and then by the second antibody would have a diameter of about 650 A, a value close to the value of 600 A found above. Samples of such cells used directly in immunofluorescence microscopy (Fig. lb) showed fluorescent fibers, with a distribution and organization identical to that reported previously (2-10, 16-18). Comparison
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Proc. Natl. Acad. Sd. USA 75 (1978)
FIG. 1. Fluorescent micrographs of 3T3 cells after glutaraldehyde fixation (a, b, and d) or after formaldehyde fixation (c). (a, b, and c) Monospecific anti-tubulin antibody. (d) Antiactin antibody. Fixation: in q and d, procedure 2; in b, procedure 1; in c, formaldehyde fixation as in ref. 20. The network of fluorescent fibers seen in b can be compared to the microtubules seen in thin sections of cells processed through the identical procedure and then viewed in the electron microscope (Fig. 2 c and d). (X950.)
of these images with the now classical images of cytoplasmic microtubules revealed after formaldehyde fixation (refs. 2-10; see also Fig. 1c) or after methanol fixation (3, 10) emphasizes
that the microtubular images seen after glutaraldehyde treatment are often more uniformly stained and are always unbroken. Experiments with procedure 2 showed that, when thin
Cell Biology: Weber et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 2. Electron micrographs of thin sections of 3T3 cells treated by procedure 1. (a) NaBH4 procedure, but no antibody treatment (steps a-d, then j). Microtubules are well preserved after the NaBH4 step, although the microtubular surface may be slightly rougher than in untreated cells. (b) NaBH4 procedure, then treatment with nonimmune IgGs followed by the second antibody (steps a-g, i, and j with nonimmune IgGs at 100 iug/ml instead of tubulin antibody in step f). Microtubular diameter (250 A) is not changed when nonimmune IgGs and the second antibody are used. (c and d) NaBH4 procedure, then treatment with monospecific antitubulin antibody followed by the second antibody (steps a-g, i, and j). Microtubular diameter is increased to approximately 600 A. Note the underlying tubule structure visible in c (arrowheads) and that intermediate filaments can be seen close to microtubules in all figures. The arrangement of microtubules in d, in which it is clear that individual microtubules can bend, can be compared to the fluorescent images seen in cells processed through step g by the identical procedure (Fig. lb). (Bars = 0.5 Am; a-c, X45,000; d, X18,000.)
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sections were examined in the electron microscope, specific decoration of microtubules also was seen. Thus, the parallel use of cells for electron microscopy and immunofluorescence microscopy documents by electron microscopic analysis that (i) normal microtubules are present and well preserved before addition of the tubulin antibody (after step d of procedure 1) (Fig. 2a); (ut) that microtubules are specifically decorated during the procedure used in immunofluorescence microscopy (compare Fig. 2 b and c); (iii) that the specifically decorated microtubules (Fig. 2c) are unbroken and must be present in those samples examined in immunofluorescence microscopy (see Fig. lb). Once again, we emphasize that there seem to be no theoretical arguments why individual microtubules cannot be detected by immunofluorescence microscopy (2, 8) and we will show elsewhere that this is indeed the case. Glutaraldehyde-fixed cells can also be used in indirect immunofluorescence microscopy with actin antibodies. Fig. 2d shows submembraneous bundles of microfilaments in 3T3 cells (1, 19) together with excellent preservation of surface detail and a more pronounced general cytoplasmic fluorescence than in similar micrographs obtained with formaldehyde-fixed cells. It is possible that glutaraldehyde-fixed cells reveal more of the cellular actin organization outside the microfilament bundles. That glutaraldehyde fixation may abolish immunological recognition in the case of other antigens or other antibodies, is not excluded. In the case of tonofilaments in rat kangaroo PtK2 cells, we observed such an example even with formaldehyde fixation (3). Microtubules are an especially suitable structure to document through a direct antibody decoration of the antigen without the use of either an enzyme product (peroxidase technique) or a density marker (ferritin technique). Clearly, in the case of shorter and smaller antigenic structures the use of peroxidase and ferritin labeling will still be necessary. Our results obtained on extensively glutaraldehyde-fixed cells support the previous report on immunoperoxidase labeling of cytoplasmic microtubules in cells fixed with very low concentrations of glutaraldehyde (13). The use of NaBH4 to reduce excess aldehyde groups may also be of value in the immunoperoxidase and immunoferritin approaches in order to suppress nonspecific binding and allow accurate diameter measurements because pretreatments with nonimmune sera or IgGs (e.g., ref. 14) may then become unnecessary. Both electron microscopic (e.g., ref. 23) and immunofluorescent studies (10) suggest that microtubular preservation is influenced by the conditions used for fixation. Because we can now correlate microtubules viewed by the two methods, it seems possible to use immunofluorescence microscopy to monitor the conditions required to obtain
Proc. Natl. Acad. Sci. USA 75 (1978)
maximal preservation of microtubules at the ultrastructural level. We appreciate the help of H. J. Koitzsch, B. Sintram, and T. Born. 1. Lazarides, E. & Weber, K. (1974) Proc. Nati. Acad. Sci. USA 71,2268-2272. 2. Weber, K., Pollack, R. & Bibring, T. (1975) Proc. Natl. Acad. Sci. USA 72,459-463. 3. Osborn, M., Franke, W. W. & Weber, K. (1977) Proc. Nati. Acad. Sci. USA 74,2490-2494. 4. Brinkley, B. R., Fuller, G. M. & Highfield, D. P. (1975) Proc. Natl. Acad. Sci. USA 72,4981-4985. 5. Osborn, M. & Weber, K. (1976). Proc. Natl. Acad. Sci. USA 73, 867-871. 6. Frankel, F. R. (1976) Proc. Natl. Acad. Sci. USA 73, 27982802. 7. Osborn, M. & Weber, K. (1976) Exp. Cell Res. 103,331-340. 8. Weber, K. (1976) in Cell Motility, eds. Goldman, R. Pollard, T. & Rosenbaum, J., (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Book A, pp. 403-417. 9. Brinkley, B. R., Fuller, G. M. & Highfield, D. P. (1976) in Cell Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Book A, pp. 435-456. 10. Osborn, M. & Weber, K. (1977) Cell 12,561-571. 11. Roberts, K. (1974) Prog. Biophys. Mol. Biol. 28,371-420. 12. Tilney, 0. G. (1971) in Origin & Continuity of Cell Organelles, eds. Beermann, W., Reinert, J. & Ursprung, H. (Springer, Heidelberg, Germany), pp. 222-256. 13. DeMey, J., Hoebeke, I., deBrabander, M., Geuens, G. & Joniau, M. (1976) Nature 264,273-275. 14. Forer, A., Kalnins, V. I. & Zimmermann, A. M. (1976) J. Cell Sci. 22, 115-131. 15. Sato, H., Ohnuki. Y. & Fujiwara, K. (1976) in Cell Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J., (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Book A, pp. 419-433. 16. Sabatini, D. D., Bensch, K. & Barrnett, R. J. (1963) J. Cell Biol. 17, 19-58. 17. Cande, W. Z., Lazarides, E. & McIntosh, J. R. (1977) J. Cell Biol.
72,552-567. 18. Weber, K., Wehland, J. & Herzog, W. (1976) J. Mol. Biol. 102, 817-829. 19. Weber, K., Rathke, P. C., Osborn, M. & Franke, W. W. (1976) Exp. Cell Res. 102, 285-297. 20. Weber, K., Bibring, T. & Osborn, M. (1975) Exp. Cell Res. 95, 111-120. 21. Franke, W. W., Lfider, M. R., Kartenbeck, J., Zerban, H. & Keenan, T. W. (1976) J. Cell Biol. 69, 173-195. 22. Peters, K. & Richards, F. M. (1977) Annu. Rev. Biochem. 46, 523-551. 23. Luftig, R. B., McMillan, P. N., Weatherbee, J. A. & Weihing, R. R. (1977) J. Histochem. Cytochem. 25, 175-187.
Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy (tubulin antibody/NaBH4/fixation procedures/actin/immunocytochemistry)
KLAUS WEBER, PETER C. RATHKE, AND MARY OSBORN Max Planck Institute for Biophysical Chemistry, D-3400 Goettingen, Federal Republic of Germany
Communicated by Manfred Eigen, January 10, 1978
ABSTRACT Electron microscopy and indirect immunofluorescence microscopy using monospecific tubulin antibodies were performed in parallel on glutaraldehyde-fixed tissue culture cells without osmium fixation. In order to reduce the excess aldehyde groups of the strongly crosslinked cellular matrix, which normally interfere with subsequent immunofluorescence microscopy, a mild NaBH4 treatment was introduced during or after the dehydration steps. Cells processed through the NaBH4 step show, in transmission electron microscopy, normal cytoplasmic microtubules ap roximately 250 A in diameter. When such cells are subjected to indirect immunofluorescence microscopy using monospecific tubulin antibody they reveal a complex system of unbroken, fine, fluorescent fibers traversing the cytoplasm between the perinuclear space and the plasma membrane. Thin sections of cells processed through the indirect immunofluorescence procedure show antibody-ecorated microtubules with a diameter of approximately 600 A. This decoration is not obtained when nonimmune IgGs are used instead of monospecific antitubulin IgGs. Thus, a direct comparison of cytoplasmic microtubules in glutaraldehyde-fixed cells by both electron microscopy and immunofluorescence microscopy can be obtained. Use of specific antibodies against actin and tubulin allows the distribution of microfilament bundles (1) and the organization of cytoplasmic microtubules (2) in tissue culture cells to be demonstrated by indirect immunofluorescence microscopy. In addition, the organization of tonofilaments has been demonstrated in one cell line by using an autoimmune serum (3). The advantages of the procedure include the direct overview provided over the whole cell and the opportunity to study numerous cells simultaneously. Documentation of cytoplasmic microtubules in tissue culture cells by immunofluorescence microscopy (2-10) has indicated the following features. (i) Microtubules are present in large numbers during interphase (2, 4). (ii) Microtubules can be followed for very long distances. They traverse the cytoplasm from the perinuclear space toward the plasma membrane and can also run for long distances underneath the plasma membrane (2, 4-10). (iii) Many of these cortical microtubules seem to originate in the centrosphere acting as an organization center, and after depolymerization they appear to regrow in a unidirectional manner toward the plasma membrane (5-7). (iv) At the onset of mitosis the complex pattern of cytoplasmic microtubules is reorganized to form the microtubules of the mitotic spindle. At late telophase, or in early G1, cytoplasmic microtubules reappear in the daughter cells originating from the centrosphere (4, 8-10). Although these results confirm and extend previous data on
microtubules obtained by conventional transmission electron microscopy on small cell samples (e.g., see refs. 11 and 12) and although the use of the immunoperoxidase technique has shown that tubulin antibodies decorate cytoplasmic microtubules as ascertained by low-power electron microscopy (13), it is desirable to examine cells processed in the same manner by both immunofluorescence and electron microscopy. This is particularly necessary because the fixation procedures traditionally used in the two techniques have been different. Indirect immunofluorescence microscopy has often been criticized because of the use of formaldehyde rather than glutaraldehyde for fixation (e.g., refs. 14 and 15). Extensive fixation with glutaraldehyde (16) is considered a requirement for microtubular preservation on the ultrastructural level, and the mild formaldehyde fixation usually performed in immunofluorescence studies (1, 2) is assumed to be accompanied by a disintegration of the delicate microtubular structure either during fixation or during dehydration (14, 15). Extensive fixation with glutaraldehyde, however, has been avoided in immunofluorescence microscopy because the background staining experienced in such studies has been severe (e.g., ref. 17). A common fixation procedure satisfying both the needs of electron microscopy and the approach used in immunofluorescence microscopy has not been obtained. Here we show that glutaraldehyde-fixed cells treated with NaBH4 and then processed through the indirect immunofluorescent procedure can be viewed in either the fluorescence or the electron microscope. Immunofluorescence microscopy shows a striking display of microtubules similar to that found after formaldehyde fixation (2-10). Electron microscopy shows microtubules specifically decorated with antibody. MATERIALS AND METHODS Cells and Antibodies. Mouse 3T3 cells were grown on round 12-mm glass coverslips (5). Antitubulin antibody-was raised in rabbits against homogeneous 6S porcine cerebrum tubulin free of microtubule-associated proteins and was made monospecific (18). The arguments for the tubulin specificity of such antibodies have been summarized (10). In addition, our antibodies can be used to measure tubulin quantitatively in a radioimmunoassay (unpublished data). The fluorescein-labeled goat antibody against rabbit IgG was from Miles Yeda, Israel, and was used after 1:10 dilution into phosphate buffered saline (Pi/NaCl). The rabbit antiactin antibody has been described
(1, 19).
Preparation of Samples. Procedure 1. (a) Fix in 2.5% glutaraldehyde (Serva, Heidelberg, F.R.G.; no. 23114) in 0.1 M Na
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: Pi/NaCl, phosphate-buffered saline.
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Cell Biology: Weber et al. cacodylate/10 mM KCI/5 mM MgCl2, pH 7.2 for 1Q0.tiwiat room temperature, followed by 10 min on ice. (b) Rinse four times, 7 min each, with ice-cold 0.1 M Na cacodylate, pH 7.2. (c) Serially dehydrate through ice-cold 50%, 70%, 80%, 90%, and 96% ethanol for 15 min each. (d) Treat with NaBH4 (Merck Co., F.R.G.), 0.5 mg/ml in 96% ethanol, three times for 6 min each at 40. (Under these conditions, NaBH4, dissolves slowly; solutions were made up 3-4 min prior to the addition of the coverslips.) Wash two times for 5 min each with 96% ethanol at 46. (e) Serially rehydrate through ethanol at 50% (40), 20%, and 10% at room temperature and equilibrate with Pi/NaCl at room temperature. (f) Add antitubulin antibody (0.05 mg/ml in Pi/NaCl) and incubate 45 min at 370; wash well with Pi/ NaCl. (g) Add fluorescein-labeled goat antirabbit antibody and incubate 45 min at 370; wash well with Pi/NaCl. (h) For immunofluorescence microscopy, coverslips were mounted in Elvanol and examined with a Zeiss fluorescence microscope equipped with epifluorescent illumination (for details, see ref. 20). (i) For electron microscopic analysis, steps a, b, and c are repeated. (j) Dehydrate the cells in water-free ethanol followed by water-free propylene oxide and embed as monolayers in Epon 812. Ultrathin sections were cut parallel to the original substratum and stained with uranyl acetate and lead citrate (for details, see ref. 21) and examined with a Philips 301 electron microscope. Procedure 1 was designed to stay as close as possible to normal electron microscopy procedures. The conditions for the NaBH4 step have not yet been optimized. Preliminary experiments suggest that the NaBH4 treatment can also be done either in step c in'50% ethanol, in which case the further serial dehydration in this step can be omitted, or in Pi/NaCl after step e using steps b and c from procedure 2. Procedure 2. (a) Fix in 1% glutaraldehyde in Pi/NaCl for 15 min at room temperature; treat with methanol at -10° for 15 min. (b) Treat with NaBH4, 0.5 mg/ml in Pi/NaCl, three times for 4 min each at room temperature. (c) Wash with Pi/NaCl two times for 3 min each at room temperature. (d) Use steps f and g of procedure 1. For fluorescence microscopy, use step h above; for electron microscopy, use step i above. RESULTS AND DISCUSSION Fixation procedure 1 allows a parallel study of cytoplasmic microtubules in tissue culture cells by both immunofluorescence microscopy and electron microscopy. Furthermore, the final product used in fluorescence microscopy can also be characterized by transmission electron microscopy. The need to accommodate strong glutaraldehyde fixation in the procedure and to avoid loss of antigenicity by too-harsh chemical treatments necessitated changes in the normal electron microscopic fixation of cells (e.g., ref. 21) as well as in the normal immunofluorescence microscopy (1, 2, 20). We have tried to keep these changes to a minimum. The following points of the procedure should be discussed. 1. We have omitted postfixation with osmium tetroxide because this treatment severely hampers subsequent decoration of microtubules in immunofluorescence microscopy. Athough omission of this step decreases the preservation of membraneous structures, microtubules are still well preserved in thin sections. 2. The fixation step uses a concentration of glutaraldehyde of 2.5% and an exposure time of 20 min as prescribed in electron microscopy procedures, rather than the formaldehyde fixation previously used in immunofluorescence microscopy (2-10, 20). Dehydration was performed as in normal electron microscopy studies.
Proc. Natl. Acad. Sci. USA 75 (1978)
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3.- To be able to use glutaraldehyde fixation in indirect immunofluorescence microscopy, we had to overcome the strong nonspecific background staining, described by others (e.g., ref. 17) and experienced also by us. This background is also found when nonimmune sera are used. Here we were helped by two observations with procedure 2 omitting steps b and c: (i) glutaraldehyde-fixed cells, as well as such cells after treatment with the nonfluorescent antibody, did not show autofluorescence; (if) when tubulin antibody followed by the second fluorescent antibody was used, decoration of cytoplasmic microtubules against a very high background was always observed. The nonspecific background staining was increased when the protein concentration of either the first or the second antibody was increased, regardless if the first antibody was from an immune serum or a nonimmune serum. These results suggested that, even after extensive washing, the glutaraldehyde-fixed cell matrix could contain excess covalently bound aldehyde groups which, by binding either the first or second antibody, are responsible for the unspecific background staining seen. This assumption, which is independent of whether glutaraldehyde fixation of proteins is performed via the normal dialdehyde or via a complex polymeric product obtained by aldol condensation (for a recent review, see ref. 22), was tested by introducing a gentle aldehyde reduction step. Experiments with procedure 2 showed that treatment of the glutaraldehyde-fixed cell matrix after the methanol step with the aldehyde reducing agent NaBH4 in buffer decreased the background staining dramatically. Cytoplasmic microtubules could readily be visualized in indirect immunofluorescence microscopy against a relatively dark background (Fig. la). Electron microscopic analysis of 3T3 cells subjected to NaBH4 treatment (Fig. 2a) showed normal microfilaments, intermediate filaments, and ribosomes. Because of the lack of osmium fixation, cellular membraneous structures exhibited a slightly decreased, and in some cases (e.g., mitochondria) an apparently reversed, contrast. Microtubules were relatively well preserved and stained, although their surfaces seemed rougher than in cells subjected to osmium fixation. The diameter of the microtubules was 250 A. 4. In order to assess the influence of rehydration and indirect antibody decoration on glutaraldehyde-fixed cells subjected to NaBH4 treatment, we studied the effect of rabbit nonimmune IgGs (100-200 ;zg/ml in Pi/NaCl) followed by fluorescent goat antirabbit IgGs (1 mg/ml in Pi/NaCl) on the preservation of cytoplasmic microtubules. Electron microscopic analysis demonstrated no structural damage to the microtubules as well as preservation of their diameter at approximately 250 A (Fig. 2b). In immunofluorescence microscopy, some residual staining, particularly of the nucleus, was observed (not shown). 5. Substitution of monospecific rabbit antitubulin IgGs (0.05 mg/ml) for nonimmune rabbit IgGs (see above) followed by treatment with fluorescent goat antirabbit IgGs gave a totally different impression when ultrathin sections were examined in the electron microscope. Although a normal display of microfilament bundles, intermediate filaments, and ribosomes was observed, all microtubules were heavily stained (Fig. 2 c and d) and had diameters of approximately 600 A rather than 250 A (see above). Because IgGs have a maximum length of approximately 100 A, a microtubule decorated around its circumference first by antitubulin antibody and then by the second antibody would have a diameter of about 650 A, a value close to the value of 600 A found above. Samples of such cells used directly in immunofluorescence microscopy (Fig. lb) showed fluorescent fibers, with a distribution and organization identical to that reported previously (2-10, 16-18). Comparison
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FIG. 1. Fluorescent micrographs of 3T3 cells after glutaraldehyde fixation (a, b, and d) or after formaldehyde fixation (c). (a, b, and c) Monospecific anti-tubulin antibody. (d) Antiactin antibody. Fixation: in q and d, procedure 2; in b, procedure 1; in c, formaldehyde fixation as in ref. 20. The network of fluorescent fibers seen in b can be compared to the microtubules seen in thin sections of cells processed through the identical procedure and then viewed in the electron microscope (Fig. 2 c and d). (X950.)
of these images with the now classical images of cytoplasmic microtubules revealed after formaldehyde fixation (refs. 2-10; see also Fig. 1c) or after methanol fixation (3, 10) emphasizes
that the microtubular images seen after glutaraldehyde treatment are often more uniformly stained and are always unbroken. Experiments with procedure 2 showed that, when thin
Cell Biology: Weber et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 2. Electron micrographs of thin sections of 3T3 cells treated by procedure 1. (a) NaBH4 procedure, but no antibody treatment (steps a-d, then j). Microtubules are well preserved after the NaBH4 step, although the microtubular surface may be slightly rougher than in untreated cells. (b) NaBH4 procedure, then treatment with nonimmune IgGs followed by the second antibody (steps a-g, i, and j with nonimmune IgGs at 100 iug/ml instead of tubulin antibody in step f). Microtubular diameter (250 A) is not changed when nonimmune IgGs and the second antibody are used. (c and d) NaBH4 procedure, then treatment with monospecific antitubulin antibody followed by the second antibody (steps a-g, i, and j). Microtubular diameter is increased to approximately 600 A. Note the underlying tubule structure visible in c (arrowheads) and that intermediate filaments can be seen close to microtubules in all figures. The arrangement of microtubules in d, in which it is clear that individual microtubules can bend, can be compared to the fluorescent images seen in cells processed through step g by the identical procedure (Fig. lb). (Bars = 0.5 Am; a-c, X45,000; d, X18,000.)
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sections were examined in the electron microscope, specific decoration of microtubules also was seen. Thus, the parallel use of cells for electron microscopy and immunofluorescence microscopy documents by electron microscopic analysis that (i) normal microtubules are present and well preserved before addition of the tubulin antibody (after step d of procedure 1) (Fig. 2a); (ut) that microtubules are specifically decorated during the procedure used in immunofluorescence microscopy (compare Fig. 2 b and c); (iii) that the specifically decorated microtubules (Fig. 2c) are unbroken and must be present in those samples examined in immunofluorescence microscopy (see Fig. lb). Once again, we emphasize that there seem to be no theoretical arguments why individual microtubules cannot be detected by immunofluorescence microscopy (2, 8) and we will show elsewhere that this is indeed the case. Glutaraldehyde-fixed cells can also be used in indirect immunofluorescence microscopy with actin antibodies. Fig. 2d shows submembraneous bundles of microfilaments in 3T3 cells (1, 19) together with excellent preservation of surface detail and a more pronounced general cytoplasmic fluorescence than in similar micrographs obtained with formaldehyde-fixed cells. It is possible that glutaraldehyde-fixed cells reveal more of the cellular actin organization outside the microfilament bundles. That glutaraldehyde fixation may abolish immunological recognition in the case of other antigens or other antibodies, is not excluded. In the case of tonofilaments in rat kangaroo PtK2 cells, we observed such an example even with formaldehyde fixation (3). Microtubules are an especially suitable structure to document through a direct antibody decoration of the antigen without the use of either an enzyme product (peroxidase technique) or a density marker (ferritin technique). Clearly, in the case of shorter and smaller antigenic structures the use of peroxidase and ferritin labeling will still be necessary. Our results obtained on extensively glutaraldehyde-fixed cells support the previous report on immunoperoxidase labeling of cytoplasmic microtubules in cells fixed with very low concentrations of glutaraldehyde (13). The use of NaBH4 to reduce excess aldehyde groups may also be of value in the immunoperoxidase and immunoferritin approaches in order to suppress nonspecific binding and allow accurate diameter measurements because pretreatments with nonimmune sera or IgGs (e.g., ref. 14) may then become unnecessary. Both electron microscopic (e.g., ref. 23) and immunofluorescent studies (10) suggest that microtubular preservation is influenced by the conditions used for fixation. Because we can now correlate microtubules viewed by the two methods, it seems possible to use immunofluorescence microscopy to monitor the conditions required to obtain
Proc. Natl. Acad. Sci. USA 75 (1978)
maximal preservation of microtubules at the ultrastructural level. We appreciate the help of H. J. Koitzsch, B. Sintram, and T. Born. 1. Lazarides, E. & Weber, K. (1974) Proc. Nati. Acad. Sci. USA 71,2268-2272. 2. Weber, K., Pollack, R. & Bibring, T. (1975) Proc. Natl. Acad. Sci. USA 72,459-463. 3. Osborn, M., Franke, W. W. & Weber, K. (1977) Proc. Nati. Acad. Sci. USA 74,2490-2494. 4. Brinkley, B. R., Fuller, G. M. & Highfield, D. P. (1975) Proc. Natl. Acad. Sci. USA 72,4981-4985. 5. Osborn, M. & Weber, K. (1976). Proc. Natl. Acad. Sci. USA 73, 867-871. 6. Frankel, F. R. (1976) Proc. Natl. Acad. Sci. USA 73, 27982802. 7. Osborn, M. & Weber, K. (1976) Exp. Cell Res. 103,331-340. 8. Weber, K. (1976) in Cell Motility, eds. Goldman, R. Pollard, T. & Rosenbaum, J., (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Book A, pp. 403-417. 9. Brinkley, B. R., Fuller, G. M. & Highfield, D. P. (1976) in Cell Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Book A, pp. 435-456. 10. Osborn, M. & Weber, K. (1977) Cell 12,561-571. 11. Roberts, K. (1974) Prog. Biophys. Mol. Biol. 28,371-420. 12. Tilney, 0. G. (1971) in Origin & Continuity of Cell Organelles, eds. Beermann, W., Reinert, J. & Ursprung, H. (Springer, Heidelberg, Germany), pp. 222-256. 13. DeMey, J., Hoebeke, I., deBrabander, M., Geuens, G. & Joniau, M. (1976) Nature 264,273-275. 14. Forer, A., Kalnins, V. I. & Zimmermann, A. M. (1976) J. Cell Sci. 22, 115-131. 15. Sato, H., Ohnuki. Y. & Fujiwara, K. (1976) in Cell Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J., (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Book A, pp. 419-433. 16. Sabatini, D. D., Bensch, K. & Barrnett, R. J. (1963) J. Cell Biol. 17, 19-58. 17. Cande, W. Z., Lazarides, E. & McIntosh, J. R. (1977) J. Cell Biol.
72,552-567. 18. Weber, K., Wehland, J. & Herzog, W. (1976) J. Mol. Biol. 102, 817-829. 19. Weber, K., Rathke, P. C., Osborn, M. & Franke, W. W. (1976) Exp. Cell Res. 102, 285-297. 20. Weber, K., Bibring, T. & Osborn, M. (1975) Exp. Cell Res. 95, 111-120. 21. Franke, W. W., Lfider, M. R., Kartenbeck, J., Zerban, H. & Keenan, T. W. (1976) J. Cell Biol. 69, 173-195. 22. Peters, K. & Richards, F. M. (1977) Annu. Rev. Biochem. 46, 523-551. 23. Luftig, R. B., McMillan, P. N., Weatherbee, J. A. & Weihing, R. R. (1977) J. Histochem. Cytochem. 25, 175-187.
