The influence of buffers during fixation on the appearance of smooth endoplasmic reticulum and glycogen in hepatocytes of normal and glycogen-depleted rats.

Liver tissue of normal and glycogen depleted rats was prepared for transmission electron microscopy by perfusion fixation and subsequent osmication in the presence of various buffers, dehydration in aethanol and embedding in epon. The use of Na/K-phosphate or Na-cacodylate to buffer glutaraldehyde led to similar appearance and distribution of SER. When Na-cacodylate was used during osmication, more SER membranes were retained but less accumulations of glycogen were found than after osmication in the presence of Na/K-phosphate. Fixation with s-collidine buffered osmium led to an easily recognisable network of SER comprising wide tubules whereas glycogen was hindered to be stained. Veronal acetate or Na-cacodylate supplemented with sucrose resulted in marked dilation and disintegration of SER. A similar effect was obtained when Na/K-phosphate or Na-cacodylate was used in hyposmolar concentration as buffer for glutaraldehyde. Liver of fasted rats or glucagon-treated rats after perfusion with Na/K-phosphate buffered glutaraldehyde and osmication in the presence of Na/K-phosphate or Na-cacodylate comprised glycogen-depleted hepatocytes which contained abundant SER membranes occupying the entire space between other organelles even in samples harvested 3 h after glucagon administration. The diversity in appearance and distribution of SER and glycogen granules, which depends to a large extend on the buffer used, suggests that SER membranes may not be sufficiently stabilized during aldehyde fixation and osmication. We thus consider it likely that large accumulations of glycogen granules are the consequence of disintegration of SER membranes during processing rather than they represent the morphologic substrate of physiological degradation of SER membranes in the course of glycogen synthesis and deposition.     

The influence of buffers during fixation on the appearance of smooth endoplasmic reticulum and glycogen in hepatocytes of normal and glycogen-depleted rats

Summary Liver tissue of normal and glycogen depleted rats was prepared for transmission electron microscopy by perfusion fixation and subsequent osmication in the presence of various buffers, dehydration in aethanol and embedding in epon. The use of Na/K-phosphate or Nacacodylate to buffer glutaraldehyde led to similar appearance and distribution of SER. When Na-cacodylate was used during osmication, more SER membranes were retained but less accumulations of glycogen were found than after osmication in the presence of Na/K-phosphate. Fixation with s-collidine buffered osmium led to an easily recognisable network of SER comprising wide tubules whereas glycogen was hindered to be stained. Veronal acetate or Na-cacodylate supplemented with sucrose resulted in marked dilation and disintegration of SER. A similar effect was obtained when Na/K-phosphate or Na-cacodylate was used in hyposmolar concentration as buffer for glutaraldehyde. Liver of fasted rats or glucagon-treated rats after perfusion with Na/K-phosphate buffered glutaraldehyde and osmication in the presence of Na/K-phosphate or Na-cacodylate comprised glycogen-depleted hepatocytes which contained abundant SER membranes occupying the entire space between other organelles even in samples harvested 3 h after glucagon administration. The diversity in appearance and distribution of SER and glycogen granules, which depends to a large extend on the buffer used, suggests that SER membranes may not be sufficiently stabilized during aldehyde fixation and osmication. We thus consider it likely that large accumulations of glycogen granules are the consequence of disintegration of SER membranes during processing rather than they represent the morphologic substrate of physiological degradation of SER membranes in the course of glycogen synthesis and deposition.     

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

Summary A modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.     

Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

Feulgen staining of rat liver fixed in different fixatives.

In the present study rat liver pieces fixed in 1) 10 per cent buffered neutral formalin, 2) 4 per cent glutaraldehyde, 3) Heidenhain's-Susa fixative and 4) Flemming's fluid, and following hydrolysis in 1-0 N HC1 at 60degreesC for varying time periods have been stained with the UV Feulgen procedure. The results of this study reveal that following hydrolysis for different time periods the tissue material fixed in formalin show the same staining pattern as those fixed in glutaraldehyde. The material fixed in Heidenhain's-Susa displays an intense Feulgen staining after two different times of hydrolysis, and that fixed in Flemming's fluid shows particular staining intensity for a prolonged time period thus indicating better preservation of DNA than in the materials fixed in the other three fixtatives.     

Potency of microwave irradiation during fixation for electron microscopy

Summary Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl₂ and MgCl₂ led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells.Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration.We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

Investigations into the structure and fixation properties of cytoplasmic lamellae in the hamster oocyte

Summary Lamellar structures have been revealed in the cytoplasm of rapidly growing hamster oocytes by glutaraldehyde fixation and by fixation in 30% ethanol followed by osmication. The structures are not preserved after osmium tetroxide either used alone or followed by glutaraldehyde; nor are they preserved by absolute ethanol, formaldehyde, glyceraldehyde, glyoxal, 2-hydroxy-adipaldehyde or potassium permanganate. Immersion in 30% ethanol followed by extraction in distilled water and fixation in glutaraldehyde and osmium tetroxide exposes the lattice-like skeletal structure of the lamellae. The lamellae are present but slightly altered after short digestion in pepsin. Longer digestion results in complete dissolution of the structures.Supported by U.S.P.H.S. Post-doctoral Fellowship 5 F2 HD-25, 190–02.I wish to thank Prof. R. E. Coupland for his continued interest in this work and for his helpful criticisms.     

Observations on the Kinetics of Uranyl Acetate and Phosphotungstic Acid Staining of Chromatin in thin Sections for Electron Microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO₄) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with gutaraldehyde only, but stain uptake is reduced following a long wash with distilled water to a level similar to that seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO₄ increases UAc uptake by a factor of about 3.

The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO₄; similar levels for final PTA uptake are also observed.

An increase in the resin content of embedded chromatin postfixed with OsO₄ is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

Corpuscular and trilaminar appearance of cultivated chick embryo fibroblast plasma membranes

Summary Electron microscope examination of chick embryo fibroblasts grown in tissue culture and fixed in a variety of osmium fixatives as well as in fixatives not containing osmium revealed a limiting cell membrane with a corpuscular substructure. A trilaminar appearance was more difficult to demonstrate in plasma membranes of cells fixed in osmium or glutaraldehyde solutions followed by osmication, while many extensive regions of membranes with this appearance were observed after fixation in buffered potassium permanganate.A study of the conditions favorable to the demonstration of a trilaminar appearance indicated that the level of focus of the electron microscope image and alteration of the intrinsic structure of the summits within the membrane played significant roles in producing this appearance.This work was aided by Research Grant GB 2129 from the National Science Foundation. Some of the equipment used was purchased with funds from the National Institutes of Health Grant 2 TI GM 326. I wish to thank Dr. Robert M. Dougherty from the Department of Microbiology who grew and supplied me with the chick embryo fibroblast cultures used in these studies, and Mrs. Ursula Feller and Miss Leslie Hammack for their technical assistance.     

Parathyroid ultrastructure after aldehyde fixation, high-pressure freezing, or microwave irradiation

Parathyroid cell variants, commonly observed in parathyroid glands fixed by immersion in glutaraldehyde, are believed to be the result of cyclic changes in the course of parathyroid hormone secretion. Immersion of bovine parathyroid glands in a mixture consisting of 1% glutaraldehyde, 1.5% formaldehyde, and 2.5% acrolein, followed by post-fixation in 1% osmium tetroxide, resulted in high uniformity with only one cell variant, whereas the same fixation procedure led to disruption of cell membranes and formation of cell variants in rat parathyroids. Parathyroid glands of both cattle and rats prepared by high-pressure quick-freezing and subsequent freeze-substitution contained only one cell variant. Excellent preservation of the ultrastructure of bovine and rat parathyroids, also exhibiting only one cell variant, was achieved by microwave irradiation in the presence of 2.5% glutaraldehyde in Na-cacodylate followed by post-fixation with OsO4 in Na-cacodylate or s-collidine, both containing Ca2+ and Mg2+. Use of the appropriate buffer, as well as osmication, is essential for successful fixation utilizing microwave energy. The main effects are considered to be heating specimens within sufficient short periods and enhancement of subsequent osmium fixation. The results support the idea, arising after examination of perfusion-fixed parathyroid tissue, that parathyroid cell variants occur during improper aldehyde fixation rather than that they express functional diversity.     

An investigation by electron microscopy of the nucleoside phosphatase activity of amphibian and mammalian erythrocytes

Amphibian and mammalian blood was washed in isotonic saline, fixed in glutaraldehyde, and then stained in the ATPase medium of Wachstein and Meisel. The blood cells were subsequently postfixed in osmium tetroxide, embedded in epoxy resins, and studied by electron microscopy. The plasma membranes of amphibian erythrocytes, from the newt Triturus cristatus and the frog Rana esculenta, were stained after incubation in media containing ATP or ADP as substrates, but were unstained after incubation in media containing AMP or sodium β-glycerophosphate. The addition of 0.001 M ouabain to ATP-containing media did not inhibit the staining of the plasma membranes, but the omission of Mg⁺⁺ ions from the medium inhibited staining. The plasma membranes of rat and rabbit erythrocytes were never stained after incubation in any of the media used.     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about one-third those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried material fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

Summary A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about onethird those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried metarial fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Ca++-induced structural changes in photoreceptor microvilli of diptera

Summary When the compound eyes of the fly Lucilia are fixed for electron microscopy with glutaraldehyde in common buffer solutions, artefactual whorls are liable to be formed from the photoreceptor microvilli. The whorls result from two factors: (i) a prolonged time interval prior to osmication, such as the overnight primary fixation or wash at 4° C commonly used in studies of compound eyes; (ii) as little as 1–2 mM Ca⁺⁺ in the primary fixative and wash solutions. Osmication after short (1 h) glutaraldehyde fixation at 4° C, or omission of Ca⁺⁺ and addition of 2 mM EGTA, prevent whorl-formation. In the tipulid fly Ptilogyna, similar artefacts are produced, but are confined to the distal zone of the microvilli that sheds during turnover.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

Potency of microwave irradiation during fixation for electron microscopy

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

Bismuth staining for light and electron microscopy.

Bismuth salts on aldehyde fixed tissue give a highly selective pattern of staining suitable for light and electron microscopy. Structures stained include the nucleolus, ribosomes, inter- and perichromatin granules, the Golgi complex beads and the outer face of the tubule doublets of mouse sperm, certain neurosecretory vesicles believed to contain biogenic amines, some junctions (some central synapses, neuromuscular junctions, tight junctions), specialized membranes such as the post acrosomal dense lamina of mouse sperm and the inner alveolar membrane of Paramecium, and a variety of structures associated with the cytoplasmic face of membranes, such as plasma membrane plaques, cleavage furrows, the leading edge of the spreading acrosome and sperm annuli.Staining is not reduced by nucleases and spot tests show no reaction between nucleic acids and bismuth under conditions similar to those used to stain tissues. However, spot tests do show strong binding of bismuth by basic proteins and by some phosphorylated molecules.It is hypothesized that bismuth reacts with cell components in two ways, distinguishable by their glutaraldehyde sensitivity. For example, staining of the nucleolus and ribosomes is blocked by glutaraldehyde but the inter- and perichromatin granules and the GC beads are unaffected. Spot tests show that basic proteins (histones, protamines, polylysine and polyargenine) and other molecules with free amino groups (5HT, tryptamine, dopamine) bind bismuth strongly, a reaction that is blocked to varying degrees by glutaraldehyde. We presume that most bismuth staining of tissues is due to reaction with amine groups and is glutaraldehyde sensitive and some may be due to guanidine groups which are less sensitive to fixation by glutaraldehyde. Organic phosphates may be the cause of the glutaraldehyde insensitive staining since ATP and some other phosphates bind bismuth in a reaction that is not blocked by glutaraldehyde.     

Osmium dependent argentaffin staining of lysosomes

Summary Lysosomes stain with the argentaffin reaction after fixation with glutaraldehyde followed by osmium tetroxide. The reaction works well both at the level of the light and electron microscope. Control experiments show that this argentaffinity is caused by reduced osmium tetroxide. No staining could be observed in freeze-dried material, in tissues fixed only with glutaraldehyde, or after bleaching of the sections with hydrogen peroxide solutions. In the electron microscope, the population of lysosomes appears heterogeneous as related to the density of silver deposits over the organelles. No correlation is found between size and argentaffinity of lysosomes. X-ray microanalysis of sections from glutaraldehyde/osmium tetroxide fixed material reveals significantly higher amounts of osmium in lysosomes, as compared to other cell organelles (e.g. peroxisomes or mitochondria). A significant peak for silver is observed in lysosomes after treatment of the sections with ammoniacal silver solution, whereas the signal for osmium is reduced. Amounts of sulphur are too low to be detected in lysosomes. It is concluded that argentaffin staining of lysosomes is an osmium dependent reaction.Parts of these results have been presented as a poster during the 20th Congress of Electron Microscopy, joint session of the Austrian Society of Electron Microscopy and the German Society of Electron Microscopy, August 23–28, 1981, Innsbruck, Austria     

Influence of calcium and magnesium containing fixatives of the ultrastructure of parathyroids

The effect of Ca²⁺ and Mg²⁺ on feline parathyroid cells during perfusion fixation with glutaraldehyde and subsequent immersion in OsO₄ was investigated. Both Ca²⁺ and Mg²⁺ may exert a stabilizing or destabilizing effect on cell membranes and on elements of the cytoskeleton. The effect depends (1) on the ion concentration, (2) on the buffer concentration and (3) on the fixative. Stabilization due to Ca²⁺ or Mg²⁺ during glutaraldehyde fixation is not altered during subsequent osmication but both cations may cause destabilization during osmication in tissue prefixed without cations. Ca²⁺ and Mg²⁺ also reduce cell volume in combination with low osmolar buffer but they prevent cells from excessive shrinkage due to high osmolar buffers. Ca²⁺ and Mg²⁺ alone or in combination reduce swelling of RER, extraction of cellular material and loss of subcellular compartments, such as secretory granules, under optimal conditions. Ca²⁺, however, provokes formation of dark (shrunken) and light (swollen) cells accompanied by loss of subcellular components when used in low concentration during osmication. Low concentrations of Mg²⁺ added to glutaraldehyde exert similar effects. Stabilization of membranes is assumed to be due to the binding capacity of Ca²⁺ and Mg²⁺ to both phospholipids and proteins. The influence of Ca²⁺ and Mg²⁺ to changes in cell volume is considered likely to be the result of ionic interaction in the cytoplasmic gel, the maintenance of cell volume being a matter of equilibrium between the swelling pressure of the cytoplasmic gel and osmotic pressure of the fixative solution.     

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.