Amyloid associated with elastin-staining laminar aggregates in the lungs of patients diagnosed with acute respiratory distress syndrome

Background

The heterogeneity of conditions underlying respiratory distress, whether classified clinically as acute lung injury (ALI) or the more severe acute respiratory distress syndrome (ARDS), has hampered efforts to identify and more successfully treat these patients. Examination of postmortem lungs among cases clinically diagnosed as ARDS identified a cohort that showed a consistent morphology at the light and electron microscope levels, and featured pathognomonic structures which we termed elastin-staining laminar structures (ELS).

Methods

Postmortem tissues were stained using the Verhoeff-Van Gieson procedure for elastic fibers, and with Congo red for examination under a polarizing microscope. Similar samples were examined by transmission EM.

Results

The pathognomonic ELS presented as ordered molecular aggregates when stained using the Verhoeff-van Gieson technique for elastic fibers. In several postmortem lungs, the ELS also displayed apple-green birefringence after staining with Congo red, suggesting the presence of amyloid. Remarkably, most of the postmortem lungs with ELS exhibited no significant acute inflammatory cellular response such as neutrophilic reaction, and little evidence of widespread edema except for focal intra-alveolar hemorrhage.

Conclusions

Postmortem lungs that exhibit the ELS constitute a morphologically-identifiable subgroup of ARDS cases. The ordered nature of the ELS, as indicated by both elastin and amyloid stains, together with little morphological evidence of inflammation or edema, suggests that this cohort of ARDS may represent another form of conformational disease. If this hypothesis is confirmed, it will require a new approach in the diagnosis and treatment of patients who exhibit this form of acute lung injury.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

Buschke-Ollendorff syndrome presenting with asymptomatic yellowish papules and leg length discrepancy: A case report

Buschke-Ollendorff syndrome (BOS) is a rare, usually benign, autosomal dominant genetic disease affecting about 0.005% globally. BOS commonly manifests with asymptomatic connective tissue nevi, sometimes with sclerotic bone lesions like osteopoikilosis or melorheostosis. However, BOS may develop severe, symptomatic complications that require surgical intervention. Here we report a 9-year-8-month girl presenting with multiple nonpruritic, nonpainful skin plaques scattered around the trunk, buttocks, and bilateral legs. She had a history of right varus foot with inadequate plantar flexion. Upon visiting, obvious leg length discrepancy (LLD) was noted. Lesional biopsy revealed increased fibroblasts within dermal collagen bundles. Verhoeff-van Gieson stain revealed scattered foci of thickened elastic fibers between collagen fibers, especially in the mid-dermis. Radiographic examination of the lower extremities showed multiple small, round-to-oval shaped, radiopaque spots on the pelvic bones, femurs, tibiae, and both feet. Hyperostosis along the long axis with “dripping candle wax” appearance was characteristic of osteopoikilosis and melorheostosis. Genetic analysis showed heterozygous point mutation in exon 1 of LEMD3 gene (c.1323C>A, p.Y441X), confirming diagnosis of BOS. Sequential and epiphyseodesis were performed to correct LLD with a favorable outcome at 2-year follow-up. BOS associated with severe bone abnormalities is rare, but orthopedic surgical intervention can provide satisfactory outcome.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

HistoChoice as an alternative to formalin fixation of undecalcified bone specimens.

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visualized more easily in HistoChoice fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Palladium chloride as a stain for elastin at the ultrastructural level.

Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.     

Immunohistochemical analysis of smooth muscle cells and volumetric density of the elastic system fibers of wild boar (Sus scrofa) penis

The purpose of the present study was to verify the smooth muscle cell distribution and elastic system fibers volumetric density (Vv) in the corpus spongiosum and corpus cavernosum of the wild boar penis. Adult wild boars (n=13) were used. The penile mid shaft fragments were fixed with 4% phosphate buffered formalin solution and/or Bouin's liquid during 24-48 h, and processed using standard histological techniques. The sections were stained with Weigert's Resorcin-Fucsin with previous oxidation. The elastic system fibers Vv was determined in 25 random fields of each fragment using M42 test system. For immunohistochemical analysis, monoclonal anti-alpha actin smooth muscle was used. The histochemical methods detected elastic system fibers in both corpus spongiosum and corpus cavernosum of all animals. The elastic fibers Vv average was 36.6%+/-0.9 for corpus spongiosum and 11.7%+/-0.5 for corpus cavernosum. Through immunocitochemistry, a small quantity of smooth muscle cells was visualized in intimate relationship with blood vessels wall. The great amount of elastic fibers and the smooth muscle cell distribution beneath the endothelium suggest that these fibers may have an important role in penile erection process in the penis of wild boars.     

Orcein, collastin and pseudo-elastica: a re-investigation of Unna's concepts.

Orcein has been recommended for identification of elastin. Since other traditional elastica stains proved to be unspecific, it was deemed of interest to determine the selectivity of orcein and to review pertinent literature. Orcein was employed as a textile dye in ancient Egypt and was used for dyeing of wool and silk until the early 20th century. It was introduced into histological technic in 1878 as a stain for cytoplasm. Unna recommended it for demonstration of elastic tissue in 1890 and retracted claims for its specifity in 1894 because orcein colored also certain collagen fibers. Unna suggested the term collastin for collagen fibers which share the affinity of elastin for acid orcein. Other orcein solutions were used as selective stains for collagen. In histochemical studies, the staining properties of resorcin-fuchsin and orcein were very similar; elastin and various collagen fibers were strongly colored. Unna's collastin is apparently identical with the pseudo-elastica described in sections stained with resorcin-fuchsin. Both dyes react with meshworks of fine fibers, embryonic, experimentally or pathologically altered collagens. It is suggested to use the term collastin, instead of pseudo-elastica, for collagenous fibers which bind the traditional elastica stains.     

Orcein,collastin and pseudo-elastica: a re-investigation of Unna's concepts

Summary Orcein has been recommended for identification of elastin. Since other traditional elastica stains proved to be unspecific, it was deemed of interest to determine the selectivity of orcein and to review pertinent literature.Orcein was employed as a textile dye in ancient Egypt and was used for dyeing of wool and silk until the early 20th century. It was introduced into histological technic in 1878 as a stain for cytoplasm. Unna recommended it for demonstration of elastic tissue in 1890 and retracted claims for its specifity in 1894 because orcein colored also certain collagen fibers. Unna suggested the term collastin for collagen fibers which share the affinity of elastin for acid orcein. Other orcein solutions were used as selective stains for collagen.In histochemical studies, the staining properties of resorcin-fuchsin and orcein were very similar; elastin and various collagen fibers were strongly colored. Unna's collastin is apparently identical with the pseudo-elastica described in sections stained with resorcin-fuchsin. Both dyes react with meshworks of fine fibers, embryonic, experimentally or pathologically altered collagens. It is suggested to use the term collastin, instead of pseudo-elastica, for collagenous fibers which bind the traditional elastica stains.     

Microwave-assisted staining procedures in routine histopathology

Summary The use and practicability of microwave-assisted staining procedures in routine histopathology over more than three years has been evaluated. A domestic microwave oven was used to speed up the following staining procedures: Haematoxylin-Eosin (for frozen sections), Romanowsky-Giemsa, Periodic acid-Schiff (PAS), Ziehl-Neelson, Papanicolaou, Feulgen and Grocott — stain on buffered formalin fixed sections or cytologic smears. These staining procedures can be made highly reproducible providing; (1) Staining vessels are placed in the same position inside the oven; (2) Accurate timing in seconds is observed. Microwave-assisted staining procedures are equal to or even superior to those of the standard methods. Staining times can be reduced to 2%–10% of the conventional staining procedures. The basic staining protocols are presented.     

THE USE OF FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC AND DPN- AND TPN-DEPENDENT DEHYDROGENASES IN MITOCHONDRIA

Brief formalin fixation in the cold prior to histochemical assay of rat liver and pancreas for various dehydrogenases has been used successfully to circumvent the structural damage and enzymatic loss to which mitochondria of frozen sections would otherwise be subject. To obtain an optimal result a single set of conditions has been devised, including fixation prior to freezing of minute (finely diced) organ blocks in graded concentrations (0.7 to 2.0 per cent) of formaldehyde in chilled (1–4°C) Hanks'' balanced salt solution, freezing at not higher than -70°C, and use of nitro-BT or, preferably, tetranitro-BT. The present histochemical study of hepatic and acinar cells indicates that not only are succinic and D-β-hydroxybutyric dehydrogenases located exclusively in the mitochondria but so are lactic, malic, and the isocitric dehydrogenases.     

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments^1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 µm in diameter³.We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 µm inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 µm which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 µm which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O₂ and the response fades after 30-40 min at hypoxia.     

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.     

Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Summary Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.Supported in part by USPHS Grant HL-32870     

Physical Constraint Finite Element Model for Medical Image Registration

Due to being derived from linear assumption, most elastic body based non-rigid image registration algorithms are facing challenges for soft tissues with complex nonlinear behavior and with large deformations. To take into account the geometric nonlinearity of soft tissues, we propose a registration algorithm on the basis of Newtonian differential equation. The material behavior of soft tissues is modeled as St. Venant-Kirchhoff elasticity, and the nonlinearity of the continuum represents the quadratic term of the deformation gradient under the Green- St.Venant strain. In our algorithm, the elastic force is formulated as the derivative of the deformation energy with respect to the nodal displacement vectors of the finite element; the external force is determined by the registration similarity gradient flow which drives the floating image deforming to the equilibrium condition. We compared our approach to three other models: 1) the conventional linear elastic finite element model (FEM); 2) the dynamic elastic FEM; 3) the robust block matching (RBM) method. The registration accuracy was measured using three similarities: MSD (Mean Square Difference), NC (Normalized Correlation) and NMI (Normalized Mutual Information), and was also measured using the mean and max distance between the ground seeds and corresponding ones after registration. We validated our method on 60 image pairs including 30 medical image pairs with artificial deformation and 30 clinical image pairs for both the chest chemotherapy treatment in different periods and brain MRI normalization. Our method achieved a distance error of 0.320±0.138 mm in x direction and 0.326±0.111 mm in y direction, MSD of 41.96±13.74, NC of 0.9958±0.0019, NMI of 1.2962±0.0114 for images with large artificial deformations; and average NC of 0.9622±0.008 and NMI of 1.2764±0.0089 for the real clinical cases. Student’s t-test demonstrated that our model statistically outperformed the other methods in comparison (p-values <0.05).     

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.     

Permanganate Fixation of Plant Cells

In an evaluation of procedures explored to circumvent some of the problems of osmium tetroxide-fixation and methacrylate embedding of plant materials, excised segments of root tips of Zea mays were fixed for electron microscopy in potassium permanganate in the following treatment variations: unbuffered and veronal-acetate buffered solutions of 0.6, 2.0, and 5.0 per cent KMnO₄ at pH 5.0, 6.0, 6.7, and 7.5, and temperatures of 2–4°C. and 22°C. After fixation the segments were dehydrated, embedded in epoxy resin, sectioned, and observed or photographed. The cells of the central region of the rootcap are described. The fixation procedures employing unbuffered solutions containing 2.0 to 5.0 per cent KMnO₄ at a temperature of 22°C. gave particularly good preservation of cell structure and all membrane systems. Similar results were obtained using a solution containing 2.0 per cent KMnO₄, buffered with veronal-acetate to pH 6.0, and a fixation time of 2 hours at 22°C. The fixation procedure utilizing veronal-acetate buffered, 0.6 per cent KMnO₄ at 2–4°C. and pH 6.7 also gave relatively good preservation of most cellular constituents. However, preservation of the plasma membrane was not so good, nor was the intensity of staining so great, as that with the group of fixatives containing greater concentrations of KMnO₄. The other fixation procedures did not give satisfactory preservation of fine structure. A comparison is made of cell structures as fixed in KMnO₄ or OsO₄.     

The infrastructure of aortic elastic fibers

Elastic fibers are composed of a central core of elastin that is amorphous and electron-lucent in conventional transmission electron micrographs and peripheral microfibrils. A complex infrastructure within the amorphous elastin of mature rat aorta is made visible by fixation and staining with a glutaraldehyde-ruthenium red mixture in phosphate buffer or osmium-ruthenium red in cacodylate buffer. The infrastructure is composed of at least two interlacing but distinct elastic structural components; a framework of circumferentially orientated microfibrils and a three-dimensional meshwork of filaments that permeate the fiber. The latter resembles a reticulum that has previously been observed in freeze-fractured and negatively stained elastin and attributed to the supramolecular organization of elastin. Microfibrils also extend from the core of the elastic fiber into the surrounding matrix where they appear to function as anchoring fibers. These observations indicate that the elastic properties of the arterial wall are an integrated function of both elastin and microfibrils.     

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.