A new approach to map transcription sites at the ultrastructural level.

We describe a new ultrastructural method for locating transcription on ultra-thin sections. The use of anti-DNA/RNA hybrid antibodies provides specific labeling on precise structures of the nuclear compartments of several cell types. All mammalian and plant material studied (HeLa cells, lymphocytes, onion root meristematic cells) showed the same pattern of labeling: fibrillar structures in the interchromatin region and discrete regions of the dense fibrillar component at the periphery of the fibrillar centers in the nucleolus. The specificity of the immunogold labeling was tested by RNAse H digestion and by pre-blocking the antibody with synthetic DNA/RNA hybrids; in both cases no gold particles were observed. This method has considerable advantages compared with current techniques, constituting a very useful tool to map transcriptionally active loci in a variety of cells.

Diamine oxidase-gold labels histamine in human mast-cell granules: a new enzyme-affinity ultrastructural method.

We developed a post-embedding ultrastructural enzyme affinity-gold technique to label histamine. Diamine oxidase (DAO)-gold complex was prepared and tested for specificity with routinely processed, Epon-embedded, histamine-rich human mast-cell granules and histamine-agar test blocks, both of which were labeled. Specificity controls included removal of staining by DAO digestion of Epon sections containing mast cells or by filtering DAO-gold over histamine-agarose beads, and a wide variety of controls (for these specificity controls) that did not abrogate mast-cell granule or histamine-agar block labeling with DAO-gold. Internal negative controls in each sample included the failure of DAO-gold to stain Type II alveolar pneumocyte lamellar bodies or altered, swollen mast-cell granules from which histamine has been released. The new post-embedding enzyme affinity-gold technique is effective in optimally prepared ultrastructural samples that allow clear identification of key ultrastructural morphology.

A One Step Indigogenic Ultrastructural Method for Na+-K+ ATPase

The use of 5-nitroindoxyl phosphate (NIP) as an indigogenic substrate for Na+ -K+ -ATPase had been reported, further investigation was necessary to simplify this procedure to a one step method. This paper reports the use of lead citrate as atraping agent in this method, and a comparison with the well-known p-nitrophenyl phosphate (NPP) method.

An ultrastructural method for the study of cell viability using ruthenium red

Ruthenium Red (RR), a colored inorganic dye, has been used in electron microscopy to stain and visualize the glycoproteins of the cell coat. It has an average particle size of 1.13 nm and does not seem to penetrate the intact plasma membrane. Consequently the cytoplasmic and nuclear components of viable cells are not stained with this electron-opaque marker.We have used these properties of RR to develop a method for the study of abnormal permeability of damaged cell membrane and to stain nonviable cells. Under certain conditions, RR stains the cytoplasm of damaged cells in suspension (Figs. 1,2) or in culture in situ (Figs. 3-5). The stained (permeable) cells are easily visualized under the light and electron microscope.Cell suspension of an ascitic Novikoff Hepatoma and cell culture from a Rhabdomyosarcoma were utilized for this study.

An ultrastructural method for the use of polyvinylpyrrolidone and dextrans as electron opaque tracers.

A simple method has been developed whereby polymers of polyvinylpyrrolidone and dextran can be visualized at the ultrastructural level by postfixing tissue with osmium tetroxide partially reduced by potassium ferrocyanide. Further enhancement of polymer electron opacity is obtained by counterstaining thin sections with bismuth subnitrate chelated by alkaline tartrate. These tracers remain adequately dispersed in serum and, depending on molecular size and concentration, are easily detected at the ultrastructural level within vascular lumena, basement lamina, apical vacuoles of proximal and distal tubules and endothelial vesicles. This technique offers the potential of studying nephron and cell permeability with families of tracers, the members of which are similar in chemical affinities, charge density and molecular shape.

Congenital neutropenia: an intrinsic cell defect demonstrated by electron microscopy of soft agar colonies

Congenital neutropenia (CN), a disease characterized by recurrent infections leading to death in infancy, shows a maturation arrest of the myeloid series at the promyelocyte-myelocyte level. The potential value of marrow transplantation in this disease would be determined by the nature of the underlying defect. However, studies to date have failed to define whether the defect is intrinsic in the cells or attributable to “environmental” factors. Therefore, marrow of four patients with CN was cultured on soft agar, and the colonies were analyzed by a newly developed ultrastructural method. In parallel, patients' cells were used in feeder layers for normal marrow. Although the patients' colonies appeared grossly normal in size and number, electron microscopy showed only rare neutrophil colonies. These colonies contained markedly aberrant cells exhibiting asynchronous nucleocytoplasmic maturation, convoluted nuclei, excessive cytoplasm, and dearth of granules. Monocyte and eosinophil colonies differentiated normally. Patients' cells and sera supported growth of normal colonies. The studies have demonstrated unequivocally that the neutrophil cell line of patients with CN is intrinsically defective and suggest that attempts at marrow grafting are warranted.

Congenital neutropenia: an intrinsic cell defect demonstrated by electron microscopy of soft agar colonies

Congenital neutropenia (CN), a disease characterized by recurrent infections leading to death in infancy, shows a maturation arrest of the myeloid series at the promyelocyte-myelocyte level. The potential value of marrow transplantation in this disease would be determined by the nature of the underlying defect. However, studies to date have failed to define whether the defect is intrinsic in the cells or attributable to “environmental” factors. Therefore, marrow of four patients with CN was cultured on soft agar, and the colonies were analyzed by a newly developed ultrastructural method. In parallel, patients' cells were used in feeder layers for normal marrow. Although the patients' colonies appeared grossly normal in size and number, electron microscopy showed only rare neutrophil colonies. These colonies contained markedly aberrant cells exhibiting asynchronous nucleocytoplasmic maturation, convoluted nuclei, excessive cytoplasm, and dearth of granules. Monocyte and eosinophil colonies differentiated normally. Patients' cells and sera supported growth of normal colonies. The studies have demonstrated unequivocally that the neutrophil cell line of patients with CN is intrinsically defective and suggest that attempts at marrow grafting are warranted.