Picrosirius red staining of cardiac muscle following phosphomolybdic acid treatment

When the picrosirius red technique was applied to cardiac muscle sections, intense yellow myocyte staining sometimes obscured thin collagenous septa. The picrosirius red technique was modified to include treatment of the sections in 0.2% (w/v) aqueous phosphomolybdic acid prior to staining. With 1-5 min treatment, cytoplasmic staining was eradicated; diminution of collagen staining occurred only with long treatments at much higher concentrations of phosphomolybdic acid. Using this phosphomolybdic acid-picrosirius red technique, collagenous septa as thin as 0.2-0.5 micron and fine collagen fibers making up the septa were clearly discernible. The technique also worked well on sections stained by other techniques and then destained. The phosphomolybdic acid-picrosirius red technique should be useful in experiments designed to investigate the effects of collagen distribution on the electrical and mechanical behavior of cardiac muscle.     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections

Synopsis Sirius Red, a strong anionic dye, stains collagen by reacting, via its sulphonic acid groups, with basic groups present in the collagen molecule. The elongated dye molecules are attached to the collagen fibre in such a way that their long axes are parallel. This parallel relationship between dye and collagen results in an enhanced birefringency.Examination of tissue sections from 15 species of vertebrates suggests that staining with Sirius Red, when combined with enhancement of birefringency, may be considered specific for collagen. An improved and modified method of staining with Sirius Red is presented.     

Picrosirius Red Staining of Dental Structures

Histologic sections of dog mandibles and teeth were stained with picrosirius red and Mayer's hematoxylin. Collagenous structures of the mandible stained brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone were seen clearly after this procedure. Under polarized light collagen fibers could be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.     

Ruthenium red staining of rat glomerulus

Summary The irregularity of ruthenium red (RR) staining for electron microscopy may be partly attributable to its poor diffusion through tissue. In this study, ruthenium red was perfused into kidneys of normal and nephrotic rats. A dense endothelial reaction occurred in both arterioles and glomerular capillaries. Dot-like deposits, most prevalent in the lamina rara externa of the basal lamina of the glomerular capillaries, were also present. The deposits appeared with equal frequency in both normal and nephrotic animals, suggesting a similar distribution of acidic glycoprotein residues. The possible significance of these deposits and other applications for the technique are discussed.     

Nile red staining of lysosomal phospholipid inclusions

Summary We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.     

Nile red staining of lysosomal phospholipid inclusions.

We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.     

Preferential staining of granulocytic cells by sulphonaphthyl red

Using sulphonaphythyl red (Michrome #947), granules in mature and immature granulocytic cells stained bright red. Granules were not visualized in other types of normal or leukemic blood cells. As such, sulphonaphthyl red may be useful in distinguishing normal and abnormal granulocytic cells from other types of blood and bone marrow cells.     

Ultrahistochemical study on the ruthenium red surface staining

Summary The cell coat picture effect which is usually obtained with the conventional RR method, that is with the RR/OsO₄ coupled reaction, is investigated. In this first paper, each of conceivable events which might take place between RR, OsO₄ and cell surface membrane is discussed or studied. Various tests are carried out on ascites Ehrlich carcinoma cells and Zajdela ascites hepatoma cells treated with numerous chemical reagents, as also on a few pure proteins. The set of data supports the concept that the staining pattern is due to the combination in surface membranes of RR with a colloidal-like form of OsO₂. The latter might occur during the formation of stable cyclic osmic acid diesters between OsO₄ and membrane unsaturated lipids. A possibility by which the resulting marker is though also to be in a colloidal-like state is put forward. A next report will deal with this problem.     

Ultrahistochemical study on the ruthenium red surface staining

Summary The electron-dense marker which is thought to produce the ruthenium red surface staining is studied. This stain is prepared under conditions which should give its rise in cell surface membrane, and its nature and charge are tested electrophoretically and by measuring the turbidity, respectively. It is a positive colloid resulting from the recharging of colloidal osmium dioxide by RR polycations. Controls on the affinity are carried out by applying positive sol to gelled agarose sections containing hyaluronic acid, polyvinyl sulfate or polylysine. Controls are also carried out on ascites Ehrlich carcinoma and Zajdela ascites hepatoma cells subjected to prior enzymatic and chemical treatments. It is found that the osmium-RR system visualizes all acidic groups in the outer hydrophilic leaflet, that is the greater part of compounds in this external cell layer. A model is presented for the mechanism underlying its rise in cell surface membrane.     

Deterioration of limestone structures associated with copper staining

Sixty limestone monuments in Central London, UK, with copper staining from brass or bronze attachments, were surveyed. Severe spalling was noted at some copper-stained sites. The survey suggests that copper initially protects the surface, but causes stone deterioration after around 70 years. Samples from four limestone monuments were analysed microbiologically and using ESEM/EDX. EDX analyses suggested the build-up of salts below a basic copper carbonate skin (salting) as the most probable explanation of the observed deterioration. Copper-stained stone was free of photosynthetic microorganisms and yielded relatively few other microorganisms. ESEM and EDX analyses of a well-conserved limestone monument surface free of copper showed the almost complete conversion of surface limestone to gypsum. The grey/brown biofilms consisted of actinomycetes and filamentous fungi. The results indicate that copper initially prevents and then enhances limestone deterioration, and that atmospheric pollutants are far less harmful than phototrophic biofilms, since surface areas completely converted to gypsum are well preserved after more than 100 years.     

Quantification of bacterial polyhydroxyalkanoic acids by Nile red staining

The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acids were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 μg Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids. Received: 13 November 1998 / Received revision: 4 February 1999 / Accepted: 12 February 1999     

Picrosirius Red Staining of Cardiac Muscle Following Phosphomolybdic Acid Treatment

When the picrosirius red technique was applied to cardiac muscle sections, intense yellow myocyte staining sometimes obscured thin collagenous septa. The picrosirius red technique was modified to include treatment of the sections in 0.2% (w/v) aqueous phosphomolybdic acid prior to staining. With 1-5 min treatment, cytoplasmic staining was eradicated; diminution of collagen staining occurred only with long treatments at much higher concentrations of phosphomolybdic acid. Using this phosphomolybdic acid-picrosirius red technique, collagenous septa as thin as 0.2-0.5 /im and fine collagen fibers making up the septa were clearly discernible. The technique also worked well on sections stained by other techniques and then destained. The phosphomolybdic acid-picrosirius red technique should be useful in experiments designed to investigate the effects of collagen distribution on the electrical and mechanical behavior of cardiac muscle.     

Subcellular localization of neutral red staining in Limulus ventral photoreceptors

Limulus ventral photoreceptors are vitally stained by neutral red. In other systems such staining has been correlated with the presence of monoamines or neuropeptides. The stained cellular components in ventral photoreceptors are clusters of small ovoids which have been identified as residual bodies. These structures are unlikely candidates for monoamine or neuropeptide synthesis or storage sites, but may be part of the cyclic synthesis and degradation of photosensitive membrane. While vital staining with neutral red is a particularly useful method for identifying certain classes of neurons in vivo, in the case of ventral photoreceptors, the association of the vital staining property with the presence of a particular class of neurotransmitter candidates has proven difficult. Neutral red is useful, however, for visualizing the segmentation of ventral photoreceptors in vivo.