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 immu- microtubules 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 antibodies 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 microtubule-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 fluorescence microscope 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