Proc. Natl. Acad. Sci. USA Vol. 75, No. 4, pp. 1820-1824, April 1978

Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy (tubulin /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 Communicated by Manfred Eigen, January 10, 1978

ABSTRACT Electron microscopy and indirect immu- obtained by conventional transmission electron nofluorescence microscopy using monospecific tubulin anti- microscopy on small cell samples (e.g., see refs. 11 and 12) and bodies were performed in parallel on glutaraldehyde-fixed tissue use of the technique has shown culture cells without osmium fixation. In order to reduce the although the immunoperoxidase excess aldehyde groups of the strongly crosslinked cellular that tubulin decorate cytoplasmic microtubules as matrix, which normally interfere with subsequent immu- ascertained by low-power electron microscopy (13), it is de- nofluorescence microscopy, a mild NaBH4 treatment was in- sirable to examine cells processed in the same manner by both troduced during or after the dehydration steps. Cells processed immunofluorescence and electron microscopy. This is partic- through the NaBH4 step show, in transmission electron mi- ularly necessary because the fixation procedures traditionally croscopy, normal cytoplasmic microtubules ap roximately 250 Indirect im- A in diameter. When such cells are subjected to indirect im- used in the two techniques have been different. munofluorescence microscopy using monospecific tubulin munofluorescence microscopy has often been criticized because antibody they reveal a complex system of unbroken, fine, fluo- of the use of formaldehyde rather than glutaraldehyde for rescent fibers traversing the cytoplasm between the perinuclear fixation (e.g., refs. 14 and 15). Extensive fixation with glutar- space and the plasma membrane. Thin sections of cells pro- aldehyde (16) is considered a requirement for microtubular cessed through the indirect immunofluorescence procedure preservation on the ultrastructural level, and the mild formal- show antibody-ecorated microtubules with a diameter of ap- dehyde fixation usually performed in immunofluorescence proximately 600 A. This decoration is not obtained when non- immune IgGs are used instead of monospecific antitubulin studies (1, 2) is assumed to be accompanied by a disintegration IgGs. Thus, a direct comparison of cytoplasmic microtubules of the delicate microtubular structure either during fixation or in glutaraldehyde-fixed cells by both electron microscopy and during dehydration (14, 15). Extensive fixation with glutaral- immunofluorescence microscopy can be obtained. dehyde, however, has been avoided in immunofluorescence microscopy because the background staining experienced in Use of specific antibodies against actin and tubulin allows the such studies has been severe (e.g., ref. 17). A common fixation distribution of microfilament bundles (1) and the organization procedure satisfying both the needs of electron microscopy and of cytoplasmic microtubules (2) in tissue culture cells to be the approach used in immunofluorescence microscopy has not demonstrated by indirect immunofluorescence microscopy. been obtained. In addition, the organization of tonofilaments has been dem- Here we show that glutaraldehyde-fixed cells treated with onstrated in one cell line by using an autoimmune serum (3). NaBH4 and then processed through the indirect immunofluo- The advantages of the procedure include the direct overview rescent procedure can be viewed in either the fluorescence or provided over the whole cell and the opportunity to study nu- the electron microscope. Immunofluorescence microscopy merous cells simultaneously. shows a striking display of microtubules similar to that found Documentation of cytoplasmic microtubules in tissue culture after formaldehyde fixation (2-10). Electron microscopy shows cells by immunofluorescence microscopy (2-10) has indicated microtubules specifically decorated with antibody. 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 MATERIALS AND METHODS from the perinuclear space toward the plasma membrane and Cells and Antibodies. Mouse 3T3 cells were grown on round can also run for long distances underneath the plasma mem- 12-mm glass coverslips (5). Antitubulin antibody-was raised in brane (2, 4-10). (iii) Many of these cortical microtubules seem rabbits against homogeneous 6S porcine cerebrum tubulin free to originate in the centrosphere acting as an organization center, of -associated proteins and was made monospecific and after depolymerization they appear to regrow in a unidi- (18). The arguments for the tubulin specificity of such anti- rectional manner toward the plasma membrane (5-7). (iv) At bodies have been summarized (10). In addition, our antibodies the onset of mitosis the complex pattern of cytoplasmic mi- can be used to measure tubulin quantitatively in a radioim- crotubules is reorganized to form the microtubules of the mi- munoassay (unpublished data). The fluorescein-labeled goat totic spindle. At late telophase, or in early G1, cytoplasmic antibody against rabbit IgG was from Miles Yeda, Israel, and microtubules reappear in the daughter cells originating from was used after 1:10 dilution into phosphate buffered saline the centrosphere (4, 8-10). (Pi/NaCl). The rabbit antiactin antibody has been described Although these results confirm and extend previous data on (1, 19). Preparation of Samples. Procedure 1. (a) Fix in 2.5% glu- The costs of publication of this article were defrayed in part by the in 0.1 M Na payment of page charges. This article must therefore be hereby marked taraldehyde (Serva, Heidelberg, F.R.G.; no. 23114) "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: Pi/NaCl, phosphate-buffered saline. 1820 Downloaded by guest on September 29, 2021 Cell Biology: Weber et al. Proc. Natl. Acad. Sci. USA 75 (1978) 1821 cacodylate/10 mM KCI/5 mM MgCl2, pH 7.2 for 1Q0.tiwiat 3.- To be able to use glutaraldehyde fixation in indirect im- room temperature, followed by 10 min on ice. (b) Rinse four munofluorescence microscopy, we had to overcome the strong times, 7 min each, with ice-cold 0.1 M Na cacodylate, pH 7.2. nonspecific background staining, described by others (e.g., ref. (c) Serially dehydrate through ice-cold 50%, 70%, 80%, 90%, 17) and experienced also by us. This background is also found and 96% ethanol for 15 min each. (d) Treat with NaBH4 (Merck when nonimmune sera are used. Here we were helped by two Co., F.R.G.), 0.5 mg/ml in 96% ethanol, three times for 6 min observations with procedure 2 omitting steps b and c: (i) glu- each at 40. (Under these conditions, NaBH4, dissolves slowly; taraldehyde-fixed cells, as well as such cells after treatment with solutions were made up 3-4 min prior to the addition of the the nonfluorescent antibody, did not show autofluorescence; coverslips.) Wash two times for 5 min each with 96% ethanol (if) when tubulin antibody followed by the second fluorescent at 46. (e) Serially rehydrate through ethanol at 50% (40), 20%, antibody was used, decoration of cytoplasmic microtubules and 10% at room temperature and equilibrate with Pi/NaCl against a very high background was always observed. The at room temperature. (f) Add antitubulin antibody (0.05 mg/ml nonspecific background staining was increased when the pro- in Pi/NaCl) and incubate 45 min at 370; wash well with Pi/ tein concentration of either the first or the second antibody was NaCl. (g) Add fluorescein-labeled goat antirabbit antibody and increased, regardless if the first antibody was from an immune incubate 45 min at 370; wash well with Pi/NaCl. (h) For im- serum or a nonimmune serum. These results suggested that, munofluorescence microscopy, coverslips were mounted in even after extensive washing, the glutaraldehyde-fixed cell Elvanol and examined with a Zeiss matrix could contain excess covalently bound aldehyde groups equipped with epifluorescent illumination (for details, see ref. which, by binding either the first or second antibody, are re- 20). (i) For electron microscopic analysis, steps a, b, and c are sponsible for the unspecific background staining seen. This repeated. (j) Dehydrate the cells in water-free ethanol followed assumption, which is independent of whether glutaraldehyde by water-free propylene oxide and embed as monolayers in fixation of proteins is performed via the normal dialdehyde or Epon 812. Ultrathin sections were cut parallel to the original via a complex polymeric product obtained by aldol condensa- substratum and stained with uranyl acetate and lead citrate (for tion (for a recent review, see ref. 22), was tested by introducing details, see ref. 21) and examined with a Philips 301 electron a gentle aldehyde reduction step. Experiments with procedure microscope. Procedure 1 was designed to stay as close as possible 2 showed that treatment of the glutaraldehyde-fixed cell matrix to normal electron microscopy procedures. The conditions for after the methanol step with the aldehyde reducing agent the NaBH4 step have not yet been optimized. Preliminary ex- NaBH4 in buffer decreased the background staining dramati- periments suggest that the NaBH4 treatment can also be done cally. Cytoplasmic microtubules could readily be visualized in either in step c in'50% ethanol, in which case the further serial indirect immunofluorescence microscopy against a relatively dehydration in this step can be omitted, or in Pi/NaCl after step dark background (Fig. la). Electron microscopic analysis of 3T3 e using steps b and c from procedure 2. cells subjected to NaBH4 treatment (Fig. 2a) showed normal Procedure 2. (a) Fix in 1% glutaraldehyde in Pi/NaCl for 15 microfilaments, intermediate filaments, and ribosomes. Because min at room temperature; treat with methanol at -10° for 15 of the lack of osmium fixation, cellular membraneous structures min. (b) Treat with NaBH4, 0.5 mg/ml in Pi/NaCl, three times exhibited a slightly decreased, and in some cases (e.g., mito- for 4 min each at room temperature. (c) Wash with Pi/NaCl chondria) an apparently reversed, contrast. Microtubules were two times for 3 min each at room temperature. (d) Use steps f relatively well preserved and stained, although their surfaces and g of procedure 1. For fluorescence microscopy, use step h seemed rougher than in cells subjected to osmium fixation. The above; for electron microscopy, use step i above. 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 RESULTS AND DISCUSSION to NaBH4 treatment, we studied the effect of rabbit nonim- Fixation procedure 1 allows a parallel study of cytoplasmic mune IgGs (100-200 ;zg/ml in Pi/NaCl) followed by fluo- microtubules in tissue culture cells by both immunofluorescence rescent goat antirabbit IgGs (1 mg/ml in Pi/NaCl) on the microscopy and electron microscopy. Furthermore, the final preservation of cytoplasmic microtubules. Electron microscopic product used in fluorescence microscopy can also be charac- analysis demonstrated no structural damage to the microtubules terized by transmission electron microscopy. The need to ac- as well as preservation of their diameter at approximately 250 commodate strong glutaraldehyde fixation in the procedure A (Fig. 2b). In immunofluorescence microscopy, some residual and to avoid loss of antigenicity by too-harsh chemical treat- staining, particularly of the nucleus, was observed (not ments necessitated changes in the normal electron microscopic shown). fixation of cells (e.g., ref. 21) as well as in the normal immu- 5. Substitution of monospecific rabbit antitubulin IgGs (0.05 nofluorescence microscopy (1, 2, 20). We have tried to keep mg/ml) for nonimmune rabbit IgGs (see above) followed by these changes to a minimum. The following points of the pro- treatment with fluorescent goat antirabbit IgGs gave a totally cedure should be discussed. different impression when ultrathin sections were examined 1. We have omitted postfixation with osmium tetroxide in the electron microscope. Although a normal display of mi- because this treatment severely hampers subsequent decoration crofilament bundles, intermediate filaments, and ribosomes was of microtubules in immunofluorescence microscopy. Athough observed, all microtubules were heavily stained (Fig. 2 c and omission of this step decreases the preservation of membraneous d) and had diameters of approximately 600 A rather than 250 structures, microtubules are still well preserved in thin sec- A (see above). Because IgGs have a maximum length of ap- tions. proximately 100 A, a microtubule decorated around its cir- 2. The fixation step uses a concentration of glutaraldehyde cumference first by antitubulin antibody and then by the sec- of 2.5% and an exposure time of 20 min as prescribed in electron ond antibody would have a diameter of about 650 A, a value microscopy procedures, rather than the formaldehyde fixation close to the value of 600 A found above. Samples of such cells previously used in immunofluorescence microscopy (2-10, 20). used directly in immunofluorescence microscopy (Fig. lb) Dehydration was performed as in normal electron microscopy showed fluorescent fibers, with a distribution and organization studies. identical to that reported previously (2-10, 16-18). Comparison Downloaded by guest on September 29, 2021 1822 Cell Biology: Weber et al. 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 that the microtubular images seen after glutaraldehyde treat- microtubules revealed after formaldehyde fixation (refs. 2-10; ment are often more uniformly stained and are always unbro- see also Fig. 1c) or after methanol fixation (3, 10) emphasizes ken. Experiments with procedure 2 showed that, when thin Downloaded by guest on September 29, 2021 Cell Biology: Weber et al. Proc. Natl. Acad. Sci. USA 75 (1978) 1823

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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 oftubulin 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.) Downloaded by guest on September 29, 2021 1824 Cell Biology: Weber et al. Proc. Natl. Acad. Sci. USA 75 (1978) sections were examined in the electron microscope, specific maximal preservation of microtubules at the ultrastructural decoration of microtubules also was seen. level. Thus, the parallel use of cells for electron microscopy and immunofluorescence microscopy documents by electron mi- We appreciate the help of H. J. Koitzsch, B. Sintram, and T. croscopic analysis that (i) normal microtubules are present and Born. well preserved before addition of the tubulin antibody (after 1. Lazarides, E. & Weber, K. (1974) Proc. Nati. Acad. Sci. USA step d of procedure 1) (Fig. 2a); (ut) that microtubules are 71,2268-2272. specifically decorated during the procedure used in immu- 2. Weber, K., Pollack, R. & Bibring, T. (1975) Proc. Natl. Acad. Sci. nofluorescence microscopy (compare Fig. 2 b and c); (iii) that USA 72,459-463. the specifically decorated microtubules (Fig. 2c) are unbroken 3. Osborn, M., Franke, W. W. & Weber, K. (1977) Proc. Nati. Acad. and must be present in those samples examined in immu- Sci. USA 74,2490-2494. nofluorescence microscopy (see Fig. lb). Once again, we em- 4. Brinkley, B. R., Fuller, G. M. & Highfield, D. P. (1975) Proc. Natl. phasize that there seem to be no theoretical arguments why Acad. Sci. USA 72,4981-4985. individual microtubules cannot be detected by immunofluo- 5. Osborn, M. & Weber, K. (1976). Proc. Natl. Acad. Sci. USA 73, rescence microscopy (2, 8) and we will show elsewhere that this 867-871. 6. Frankel, F. R. (1976) Proc. Natl. Acad. Sci. USA 73, 2798- is indeed the case. 2802. Glutaraldehyde-fixed cells can also be used in indirect im- 7. Osborn, M. & Weber, K. (1976) Exp. Cell Res. 103,331-340. munofluorescence microscopy with actin antibodies. Fig. 2d 8. Weber, K. (1976) in Cell Motility, eds. Goldman, R. Pollard, T. shows submembraneous bundles of microfilaments in 3T3 cells & Rosenbaum, J., (Cold Spring Harbor Laboratory, Cold Spring (1, 19) together with excellent preservation of surface detail and Harbor, NY), Book A, pp. 403-417. a more pronounced general cytoplasmic fluorescence than in 9. Brinkley, B. R., Fuller, G. M. & Highfield, D. P. (1976) in Cell similar micrographs obtained with formaldehyde-fixed cells. Motility, eds. Goldman, R., Pollard, T. & Rosenbaum, J. (Cold It is possible that glutaraldehyde-fixed cells reveal more of the Spring Harbor Laboratory, Cold Spring Harbor, NY), Book A, cellular actin organization outside the microfilament bundles. pp. 435-456. 10. Osborn, M. & Weber, K. (1977) Cell 12,561-571. That glutaraldehyde fixation may abolish immunological 11. Roberts, K. (1974) Prog. Biophys. Mol. Biol. 28,371-420. recognition in the case of other antigens or other antibodies, is 12. Tilney, 0. G. (1971) in Origin & Continuity of Cell Organelles, not excluded. In the case of tonofilaments in rat kangaroo PtK2 eds. Beermann, W., Reinert, J. & Ursprung, H. (Springer, cells, we observed such an example even with formaldehyde Heidelberg, Germany), pp. 222-256. fixation (3). 13. DeMey, J., Hoebeke, I., deBrabander, M., Geuens, G. & Joniau, Microtubules are an especially suitable structure to document M. (1976) Nature 264,273-275. through a direct antibody decoration of the antigen without the 14. Forer, A., Kalnins, V. I. & Zimmermann, A. M. (1976) J. Cell Sci. use of either an enzyme product (peroxidase technique) or a 22, 115-131. density marker (ferritin technique). Clearly, in the case of 15. Sato, H., Ohnuki. Y. & Fujiwara, K. (1976) in Cell Motility, eds. structures the use of Goldman, R., Pollard, T. & Rosenbaum, J., (Cold Spring Harbor shorter and smaller antigenic peroxidase Laboratory, Cold Spring Harbor, NY), Book A, pp. 419-433. and ferritin labeling will still be necessary. Our results obtained 16. Sabatini, D. D., Bensch, K. & Barrnett, R. J. (1963) J. Cell Biol. on extensively glutaraldehyde-fixed cells support the previous 17, 19-58. report on immunoperoxidase labeling of cytoplasmic mi- 17. Cande, W. Z., Lazarides, E. & McIntosh, J. R. (1977) J. Cell Biol. crotubules in cells fixed with very low concentrations of glu- 72,552-567. taraldehyde (13). The use of NaBH4 to reduce excess aldehyde 18. Weber, K., Wehland, J. & Herzog, W. (1976) J. Mol. Biol. 102, groups may also be of value in the immunoperoxidase and 817-829. immunoferritin approaches in order to suppress nonspecific 19. Weber, K., Rathke, P. C., Osborn, M. & Franke, W. W. (1976) binding and allow accurate diameter measurements because Exp. Cell Res. 102, 285-297. nonimmune sera or ref. 20. Weber, K., Bibring, T. & Osborn, M. (1975) Exp. Cell Res. 95, pretreatments with IgGs (e.g., 14) may 111-120. then become unnecessary. Both electron microscopic (e.g., ref. 21. Franke, W. W., Lfider, M. R., Kartenbeck, J., Zerban, H. & 23) and immunofluorescent studies (10) suggest that microtu- Keenan, T. W. (1976) J. Cell Biol. 69, 173-195. bular preservation is influenced by the conditions used for 22. Peters, K. & Richards, F. M. (1977) Annu. Rev. Biochem. 46, fixation. Because we can now correlate microtubules viewed 523-551. by the two methods, it seems possible to use immunofluores- 23. Luftig, R. B., McMillan, P. N., Weatherbee, J. A. & Weihing, R. cence microscopy to monitor the conditions required to obtain R. (1977) J. Histochem. Cytochem. 25, 175-187. Downloaded by guest on September 29, 2021