Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.     

Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules

In the accompanying paper (Bloom, G.S., T.A. Schoenfeld, and R.B. Vallee, 1983, J. Cell Biol. 98:320-330), we reported that microtubule-associated protein 1 (MAP 1) from brain comprises multiple protein species, and that the principal component, MAP 1A, can be detected in both neuronal and glial cells by immunofluorescence microscopy using a monoclonal antibody. In the present study, we sought to determine the cellular and subcellular distribution of MAP 1A in commonly used cultured cell systems. For this purpose we used immunofluorescence microscopy and immunoblot analysis with anti-MAP 1A to examine 18 types of mammalian cell cultures. MAP 1A was detected in every culture system examined. Included among these were cells of mouse, rat, Chinese hamster, Syrian hamster, Potoroo (marsupial), and human origin derived from a broad variety of tissues and organs. Anti-MAP 1A consistently labeled mitotic spindles and stained cytoplasmic fibers during interphase in most of the cultures. These fibers were identified as microtubules by co-localization with tubulin in double-labeling experiments, by their disappearance in response to colchicine or vinblastine, and by their reorganization in response to taxol. The anti-MAP 1A stained microtubules in a punctate manner, raising the possibility that MAP 1A is located along microtubules at discrete foci that might represent sites of interaction between microtubules and other organelles. Verification that MAP 1A was, indeed, the reactive material in immunofluorescence microscopy was obtained from immunoblots. Anti-MAP 1A stained a band at the position of MAP 1A in all cultures examined. These results establish that MAP 1A, a major MAP from brain, is widely distributed among cultured mammalian cells both within and outside of the nervous system.     

Identification of a 100 kD protein associated with microtubules, intermediate filaments and coated vesicles in cultured cells

We have obtained several hybridoma clones producing antibodies to microtubule-associated proteins (MAPs) from bovine brain. Interaction of one of these antibodies, named RN 17, with cultured cells was studied by indirect immunofluorescence and immunoelectron microscopy. RN 17 antibody recognized both high molecular weight (HMW) MAPs, MAP 1 and MAP 2, in immunoblotting reaction with brain microtubules. In lysates of cultured cells, it bound to a protein doublet with a molecular weight of 100 kD. By immunofluorescence microscopy we showed that RN 17 antibody stained cytoplasmic fibrils, mitotic spindles and small particles in the cytoplasm of various cultured cells. The cytoplasmic fibrils were identified as both microtubules and intermediate filaments by double fluorescence microscopy and by their response to colcemid and 0.6 M KCl. This identification was confirmed by immunoelectron microscopy which also showed that the particles stained by RN 17 antibody are coated vesicles. Thus, cultured non-neural cells may contain a novel protein that binds to microtubules, intermediate filaments, and coated vesicles.     

Identification of a minus end-specific microtubule-associated protein located at the mitotic poles in cultured mammalian cells.

A mitosis-specific centrosomal component was studied with a human autoantibody, SP-H, which immunostained mitotic poles and interphase nuclei, and a single polypeptide with an apparent molecular mass of 200 to 230 kDa in various lines of cultured cells. Early mitotic PtK1 cells treated with 10 micrograms/ml taxol contained short bundles of parallel microtubules around the nuclei and cell periphery. At the time of nuclear envelope breakdown, the nuclear staining by SP-H disappeared, and the antigen relocated at one end of the parallel microtubules. Determination of the microtubule polarity demonstrated that the peripheral bundles of microtubules were arranged with their minus ends directed to the cell periphery, and the SP-H antigen was specifically localized at this end. Parallel microtubules were further rearranged first into a fan-like shape, and then into completely radial structures as observed by De Brabander et al. (Int. Rev. Cytol. 101, 215-274 (1986)). The SP-H antigen was always detected at the minus end domain of such microtubule-containing structures during the transformation process. When microtubules were depolymerized by nocodazole treatment, the SP-H antigen appeared as discrete cytoplasmic foci, suggesting that the antigen may self-associate, forming multimeric structures. The antigen in mitotic HeLa cell extracts co-sedimented in vitro with exogenous brain microtubules. The microtubule-associated SP-H antigen was insensitive to ATP extraction, but was removed from microtubules by treatment with 0.5 M NaCl. Thus the 200 to 230 kDa centrosomal component could be a novel microtubule-associated protein with affinity for the minus end of microtubules, and it might play an essential role in the organization of spindle poles during mitosis.     

A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.     

Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells

A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.     

Type I protein kinase a is localized to interphase microtubules and strongly associated with the mitotic spindle

We show here that type I protein kinase A is localized to microtubules during the entire cell cycle in epithelial (hepatoma, cervical carcinoma) and nonepithelial (myoblast) cell lines. The association of the type Ialpha regulatory subunit is very strong in all phases of mitosis, from prophase to cytokinesis. In interphase, the association appears weaker, reflecting perhaps a more dynamic molecular interaction. This regulatory subunit appears to recruit catalytic subunits as the latter are also associated with microtubules. BW1J hepatoma cells, stably transfected with either wild-type or mutant Ialpha regulatory subunit, are enriched in aberrant mitoses with multipolar spindles and in mono- or multinucleated giant cells. This suggests that type I protein kinase A could have a role in centrosome duplication and/or segregation, sister chromatid separation, or cytokinesis.     

Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells

The classification of MAP 2 as a microtubule-associated protein is based on its affinity for microtubules in vitro and its filamentous distribution in cultured cells. We sought to determine whether MAP 2 is also able to bind in situ to organelles other than microtubules. For this purpose, primary cultures of rat brain cells were stained for immunofluorescence microscopy with a rabbit anti-MAP 2 antibody prepared in our laboratory, as well as with antibodies to vimentin, an intermediate filament protein, and to tubulin, the major subunit of microtubules. MAP 2 was present on cytoplasmic fibers in neurons and in a subpopulation of the flat cells present in the cultures. Our observations were concentrated on the flat cells because of their suitability for high-resolution immunofluorescence microscopy. Double antibody staining revealed co-localization of MAP 2 with both tubulin and vimentin in the flat cells. Pretreatment of the cultures with vinblastine resulted in the redistribution of MAP 2 into perinuclear cables that contained vimentin. Tubulin paracrystals were not stained by anti-MAP 2. In cells extracted with digitonin, the normal fibrillar distribution of MAP 2 was resistant to several treatments (PIPES buffer plus 10 mM Ca++, phosphate buffer at pH 7 or 9) that induced depolymerization of microtubules, but not intermediate filaments. Staining of the primary brain cells was not observed with preimmune serum nor with immune serum adsorbed prior to use with pure MAP 2. We detected MAP 2 on intermediate filaments not only with anti-MAP 2 serum, but also with affinity purified anti-MAP 2 and with a monoclonal anti-MAP 2 prepared in another laboratory. We conclude from these experiments that material recognized by anti-MAP 2 antibodies associates with both microtubules and intermediate filaments. We propose that one function of MAP 2 is to cross-link the two types of cellular filaments.     

Gamma-tubulin distribution in interphase and mitotic cells upon stabilization and depolymerization of microtubules

Indirect immunofluorescence and digital videomicroscopy were used to study gamma-tubulin distribution in normal mitotic and interphase HeLa cells and after their treatment with microtubule-stabilizing (taxol) and depolymerizing (nocodazole) drugs. In interphase HeLa cells, the affinity-purified antibodies against gamma-tubulin and monoclonal antibodies against acetylated tubulin stain one or two neighboring dots, centrioles. The gamma-tubulin content in two centrioles from the same cell differs insignificantly. Mitotic poles contain fourfold amount of gamma-tubulin as compared with the centrioles in interphase. The effect of nocodazole (5 microg/ml) on interphase cells resulted in lowering the amount of gamma-tubulin in the centrosome, and in 24 h it was reduced by half. Treatment with nocodazole for 2 h caused a fourfold decrease in the gamma-tubulin content in mitotic poles. Besides, the mitotic poles were unevenly stained, the fluorescence intensity in the center was lower than at the periphery. Upon treatment with taxol (10 microg/ml), the gamma-tubulin content in the interphase cell centrosome first decreased, then increased, and in 24 h it doubled as compared with control. In the latter case, bright dots appeared in the cell cytoplasm along the microtubule bundles. However, after 24 h treatment with taxol, the total amount of intracellular gamma-tubulin did not change. Treatment with taxol for 2-4 h halved the gamma-tubulin content in the centrosome as compared with normal mitosis. In some cells, antibodies against gamma-tubulin revealed up to four microtubule convergence foci. Other numerous microtubule convergence foci were not stained. Thus, the existence of at least three gamma-tubulin pools is suggested: (1) constitutive gamma-tubulin permanently associated with centrioles irrespective of the cell cycle stage and of their ability to serve as microtubule organizing centers; (2) gamma-tubulin unstably associated with the centrosome only during mitosis; (3) cytoplasmic gamma-tubulin that can bind to stable microtubules.     

The smallest active fragment of microtubule-associated protein 4 and its interaction with microtubules in phosphate buffer

To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.     

Identification of the microtubule-associated protein map2 and its binding sites on metaphase chromosomes from cultured cells

The microtubule-associated protein MAP₂, which binds preferentially to AT sequences of DNA, can bind to metaphase chromosomes. The binding pattern of MAP₂ to chromosomes is similar to that found for the binding of the bisbenzimidazole derivative 33258 Hoechst, which also binds preferentially to AT-rich regions.     

Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins.

In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations.     

Indirect immunofluorescent staining of the microtubules in interphase and mitotic amoebae of the myxomycetePhysarum polycephalum

Summary The microtubules ofPhysarum amoebae have been decorated with rat antibodies against yeast tubulin. The indirect fluorescent staining observed in interphase amoebae and in flagellated amoebae is consistent with the three-dimensional reconstructions previously deduced from electron microscopic studies. Mitotic amoebae exhibit a pattern of fluorescence which is similar to that exhibited by mammalian cells and is consistent with the previous electron microscopic studies, except that we also observe pole-pole microtubule fibers during metaphase and anaphase and the presence of a typical midbody during cytokinesis. The various types of tripolar mitosis which are observed suggest that there is a regulatory mechanism allowing the formation of pseudo-bipolar mitotic apparatuses in amoebae possessing more than two mitotic centers during mitosis. The mitotic center, located in the middle of the centrosphere, is not fluorescent after staining of the monoasters induced with taxol suggesting the absence of tubulin in the mitotic center.     

Plant RanGAPs are localized at the nuclear envelope in interphase and associated with microtubules in mitotic cells

In animals and yeast, the small GTP-binding protein Ran has multiple functions - it is involved in mediating (i) the directional passage of proteins and RNA through the nuclear pores in interphase cells; and (ii) the formation of spindle asters, the polymerization of microtubules, and the re-assembly of the nuclear envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation by the RanGTPase protein RanGAP. Here we report the complementation of the yeast RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding RanGAP-like proteins. Confocal laser microscopy of Arabidopsis plants overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that the fusion protein is localized to patchy areas at the nuclear envelope of interphase cells. In contrast, the cellular distribution of RanGAPs in synchronized tobacco cells undergoing mitosis is characteristically different. Double-immunofluorescence shows that RanGAPs are co-localized with spindle microtubules during anaphase, with the microtubular phragmoplast and the surface of the daughter nuclei during telophase. Co-assembly of RanGAPs with tubulin correlates with these in vivo observations. The detected localization pattern is consistent with the postulated function of plant RanGAPs in the regulation of nuclear transport during interphase, and suggests a role for these proteins in the organization of the microtubular mitotic structures.     

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115?kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115?kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.     

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.     

Interactions of tobacco microtubule-associated protein MAP65-1b with microtubules

Tobacco microtubule associated protein (MAP65) (NtMAP65s) constitute a family of microtubule-associated proteins with apparent molecular weight around 65 kDa that collectively induce microtubule bundling and promote microtubule assembly in vitro. They are associated with most of the tobacco microtubule arrays in situ. Recently, three NtMAP65s belonging to the NtMAP65-1 subfamily have been cloned. Here we investigated in vitro the biochemical properties of one member of this family, the tobacco NtMAP65-1b. We demonstrated that recombinant NtMAP65-1b is a microtubule-binding and a microtubule-bundling protein. NtMAP65-1b has no effect on microtubule polymerization rate and binds microtubules with an estimated equilibrium constant of dissociation (K(d)) of 0.57 micro m. Binding of NtMAP65-1b to microtubules occurs through the carboxy-terminus of tubulin, as NtMAP65-1b was no longer able to bind subtilisin-digested tubulin. In vitro, NtMAP65-1b stabilizes microtubules against depolymerization induced by cold, but not against katanin-induced destabilization. The biological implications of these results are discussed.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Summary Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.