Phosphotungstic Acid-Chromic Acid as a Selective Electron-Dense Stain for Plasma Membranes of Plant Cells

A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO₃) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO₄ postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO₃ mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.     

A Self-Limiting, Highly Selective Chromium-Hematoxylin Stain

A compound, which is probably a cationic chelate, can be isolated as a dry powder from a hematoxylin-chrome alum lake. In aqueous acid solution this compound is an excellent nuclear stain which is extremely selective, very resistant to acids and alcohols, and self-limiting. Staining time may vary from 20 min to 16 hr without causing significant differences in staining intensity. To prepare the dry stain, dissolve 10 gm of hematoxylin, 10 gm of NaOH and 70 gm of chrome alum in 600 ml of distilled water, boil 20 min, cool and filter, allowing the filtrate to drop into 3.5 liters of absolute alcohol. Filter off the precipitate formed in the alcohol, and air dry it at room temperature. The staining solution is prepared by dissolving 3 gm of the dried precipitate in 100 ml of 3% HCl.     

A Selective Stain for Elastic Tissue (Orcinol-New Fuchsin)

A selective stain for elastic tissue (designated orcinol-new fuchsin) is described. Two grams of new fuchsin (C.I. No. 678) and 4 gm of orcinol (highest purity) are added to 200 ml of distilled water and the solution boiled for 5 min. Then 25 ml ferric chloride solution (U.S.P. IX) are added and the solution is boiled 5 min longer. The precipitate is collected and dissolved in 100 ml 95% ethanol. This is the staining solution. Sections are deparaffinized and brought to absolute ethanol, stained for 15 min at 37 °C with orcinol-new fuchsin, differentiated for 15 min in 70% ethanol, dehydrated, cleared and covered as usual.     

A Selective Stain for Eosinophils Using Two Oxazine Dyes Applied Sequentially

A methanolic solution of the oxazine textile dye, C. I. basic blue 122, followed by an aqueous alkaline solution of the oxazine dye, C. I. basic blue 141, and a brief rinse in an acetate buffer at pH 3.45 produces intense black staining of eosinophil granules. This staining was selective for eosinophils while other types of peripheral blood leukocytes showed little if any staining under the same conditions. This staining procedure may be useful for detecting eosinophils in samples of blood, bone marrow, or urine when eosinophiluria results from interstitial nephritis.     

A Selective Stain for Mitotic Figures, Particularly in the Developing Brain

A selective stain for mitotic figures is valuable where autoradiographic counting is not required, especially in the developing brain. Most work in this field has been based on conventional nuclear stains which do not differentiate mitotic figures from resting cells by color. Hematoxylin, Feulgen, gallocyanin and Nissl methods have been used particularly. The method described uses a modified Bouin fixative, followed by hydrolysis in 1 N HCl. Mitotic figures are selectively stained using crystal violet, with nuclear fast red as the counterstain for resting cells. The method has been tested using material from postnatal and fetal sheep, guinea pig and rat. Using paraffin mounted serial sections it is applicable to all organs. The method was very successful on developing rat brain, particularly for detail and quantitative estimation in the early stages of prenatal development, which was of primary interest. Nucleated cells of the erythrocytic series, keratin and what appear to be mast cells were found to stain. When nuclear counting or cell recognition were required these did not cause any difficulty, except in prenatal liver. The highly selective method presented stains mitotic figures, in all tissue tested, an intense blue against a background of red resting cells.     

The Bi-Functional Organization of Human Basement Membranes

The current basement membrane (BM) model proposes a single-layered extracellular matrix (ECM) sheet that is predominantly composed of laminins, collagen IVs and proteoglycans. The present data show that BM proteins and their domains are asymmetrically organized providing human BMs with side-specific properties: A) isolated human BMs roll up in a side-specific pattern, with the epithelial side facing outward and the stromal side inward. The rolling is independent of the curvature of the tissue from which the BMs were isolated. B) The epithelial side of BMs is twice as stiff as the stromal side, and C) epithelial cells adhere to the epithelial side of BMs only. Side-selective cell adhesion was also confirmed for BMs from mice and from chick embryos. We propose that the bi-functional organization of BMs is an inherent property of BMs and helps build the basic tissue architecture of metazoans with alternating epithelial and connective tissue layers.     

Thiosemicarbazide Used After Periodic Acid Makes Methenamine Silver Staining of Renal Glomerular Basement Membranes Faster and Cleaner

The periodic acid-methenamine silver staining technique, which is frequently used for demonstrating the renal glomerular basement membrane, requires a high degree of skill, and in some cases it may be difficult to obtain a good result. To overcome such difficulty and inconsistency, we have improved the method by performing methenamine silver staining after oxidation with periodic acid and subsequent application of thiosemicarbazide. In this procedure, this semicarbazide enhanced the reaction of methenamine silver with the glomerular basement membrane and the reaction was completed within a shorter time in comparison with the conventional method. This modification also eliminated any nonspecific reaction with the surface of the glass slide and the solution container and yielded excellent and reproducible results irrespective of the fixation method and material employed. It was also. found to stain the renal glomerular basement membrane of rabbits, which is demonstrable only with difficulty by the conventional method.     

Basement Membranes: Cell Scaffoldings and Signaling Platforms

Basement membranes are widely distributed extracellular matrices that coat the basal aspect of epithelial and endothelial cells and surround muscle, fat, and Schwann cells. These extracellular matrices, first expressed in early embryogenesis, are self-assembled on competent cell surfaces through binding interactions among laminins, type IV collagens, nidogens, and proteoglycans. They form stabilizing extensions of the plasma membrane that provide cell adhesion and that act as solid-phase agonists. Basement membranes play a role in tissue and organ morphogenesis and help maintain function in the adult. Mutations adversely affecting expression of the different structural components are associated with developmental arrest at different stages as well as postnatal diseases of muscle, nerve, brain, eye, skin, vasculature, and kidney.The basement membrane (basal lamina) was first described in muscle as a “membranaceous sheath of the most exquisite delicacy” (Bowman 1840). Microscopists subsequently identified basement membranes in nearly all tissues. In the late 1970s, the discovery of the basement membrane-rich EHS tumor led to the isolation of abundant quantities of laminin, type IV collagen, nidogen (entactin), and perlecan, enabling elucidation of their biochemical and cell-interactive properties and opening a door to an understanding of structure and function of basement membranes at a molecular level.Basement membranes are layered cell-adherent extracellular matrices (ECMs) that form part of tissue architecture, contributing both to embryonic differentiation and the maintenance of adult functions. They are evolutionarily ancient structures, likely appearing when organized communities of animal cells first emerged. These matrices serve as an extension of the plasma membrane, protecting tissues from disruptive physical stresses, and provide an interactive interface between cell and surrounding environment that can mediate local and distant signals within and between these compartments. Such signals appear to be largely processed through integrins, growth factor interactions, and dystroglycan. Basement membrane-dependent functions include the promotion of strong epidermal/dermal attachment, stabilization of the skeletal muscle sarcolemma, selectivity of glomerular filtration, and establishment of epithelial and glial cell polarization. Assembly of a functionally active basement membrane depends on the binding interactions among the large carbohydrate-modified proteins, each consisting of an array of distinct domains with unique binding properties. These components, in turn, are organized into higher ordered supramolecular assemblies that engage cell surface receptors in a developmentally and tissue-specific manner. In this review models of structure will be related to those of function based on a consideration of morphological, biochemical, cell biological, developmental, and genetic information.     

Azophloxine Ga, A Selective Stain for Light and Fluorescence Microscopy of Myoepithelial Cells

Paraffin sections from human lingual glands fixed in Carnoy's fluid No. 2 were dewaxed, hydrated and treated as follows: Mayer's acid hemalum, 5-10 min; running water, 15 min; 5% aqueous tannic acid, 10 min; distilled water, 3 changes; 1% aqueous phosphomolybdic acid, 10 min; distilled water, 3 changes; azophloxin GA, 2 gm in 9:1 methanol-acetic acid (mixed 16-20 hr before use), 5 min; 9:1 methanol-acetic acid, 2 changes; absolute alcohol, 1 dip; and apply a cover with nonfluorescent medium. Myoepithelial cells and muscle fibers were stained deep red; connective tissue fibers and serous cells, yellow; mucous cells, unstained. Only myoepithelial cells and muscle fibers were strongly fluorescent. This selective fluorescence greatly facilitated study of very fine fibers in myoepithelial cells and of the basket-like meshworks. This stain does not require differentiation and is useful in general histopathology. No fading was observed in sections stored for 1 yr.     

The Peracetic-Orcein-Halmi Stain: A Stain for Connective Tissues

A selective stain useful for the study of connective tissues is described. The stain demonstrates elastic and oxytalan fibers as well as fibrils in mucous connective tissues previously undescribed. Reticular fibers are not stained. The stain may be used on sections that have been fresh frozen or fixed in formalin or ethanol. Sections are deparaffinized, washed in absolute ethanol, oxidized in peracetic acid 30 min, washed in running water, stained in Taenzer-Unna orcein 15 min, 37°C, differentiated in 70% ethanol, washed in running water, stained in Lillie-Mayer alum hematoxylin 4 min, blued in running water, and counterstained 20 sec in a modified Halmi mixture of 100 ml distilled water, 0.2 gm light green SF, 1.0 gm orange G, 0.5 gm phosphotungstic acid and 1.0 ml glacial acetic acid. Sections are rinsed briefly in 0.2% acetic acid in 95% ethanol, dehydrated and mounted.     

Basic Fuchsin and Picro-Indigo Carmine: A Selective Stain for Cells in Mitosis and for Autoradiography

Selective staining of dividing nuclei is accomplished as follows: paraffin sections, after hydration, are stained 15 min in a saturated aqueous solution of basic fuchsin, washed, then stained 1.5 min in an equal-volumes mixture of indigo carmine saturated in 70% alcohol, and saturated aqueous picric acid. Removal of excess dye with 3 changes of 70% alcohol, dehydration, clearing and covering in a resinous medium completes the process. Nuclei of dividing cells are stained red; cytoplasm and interphase nuclei, light green. This method has been used successfully for determining the mitotic activity of skin, kidney, liver and other rabbit and mouse tissues. Tissue sections previously prepared as autoradiographs may be stained by this method to facilitate the determination of radioactive labeling of mitotic cells.     

Rhodanile Blue; a Rapid and Selective Stain for Heinz Bodies

Equal volumes of heparinized or EDTA-treated blood and a 0.5% solution of rhodanile blue (E. Gurr, Michrome No. 1156) in 1% NaCl were mixed and allowed to stand for 2 rain. Thin smears were then prepared, air dried and examined under oil. Heinz bodies stained deep purple and contrasted well with the yellow-orange to blue-green cytoplasm. Durable mounts could be made by applying a cover glass with a resinous mediiun to the dry smear (D. P. X. was used). The reticular material in reticulocytes did not stain in 2 min but could be stained by allowing the stain to act 5 min.     

A One-Solution Stain for Spermatozoa

Fresh semen is allowed to liquefy 30-60 minutes and thin, even smears of it made on clean slides or cover glasses. The smears are fixed 3 minutes with an equal-parts mixture of alcohol and ether, then air dried. They are stained 5-7 minutes in an aqueous solution made by mixing 2 volumes of 5% aniline blue (water soluble), 1 volume of 5% eosin B and 1 volume of 1% phenol. Staining at 40-60°C. is recommended. After staining, the smears are washed with distilled water, air dried and mounted in balsam or synthetic resin. The method was used on over 2000 samples of dog semen and some human specimens. Good preservation and differentiation of cytological structures was obtained uniformly, but tests were not made with other species.     

A Trichrome Stain for Insect Material

Deparaffinized insect sections are brought down to water and overstained in a 0.1% solution of azocarmine G in 1% acetic acid. They are then destained in a saturated solution of orange G until the azocarmine G is removed from the endocuticle and the latter is colored pale yellow. After washing, the sections are transferred to a 5.0 % solution of phosphotungstic acid in water for 3 min. They are then rinsed in distilled water and stained in a 0.1% solution of methyl green in 1% acetic acid until the endocuticle is green. Differentiation is done in 2 changes of 95% alcohol. The sections are then dehydrated either in absolute alcohol or dioxane, cleared in a mixture of “camsal”, eucalyptol, dioxane, and paraldehyde (1:2:2:1), and mounted in Mohr and Wehrle's medium, a mountant of the Euparal type.