DNA fiber-FISH staining mechanism.

Fluorescence in situ hybridization to DNA fibers (Fiber-FISH) is a high-resolution, wide-ranging physical DNA mapping method that finds increasing application in the study of pathological gene rearrangements. Here we present experiments designed to understand the nature of the discontinuous FISH signal patterns seen after Fiber-FISH. Use of a novel cisplatin-based chemical labeling method enabled us to produce intact biotin-labeled cosmid target DNA molecules. We monitored by immunofluorescence the fate of such cosmid targets during denaturation and hybridization. The same cosmid DNA labeled with digoxigenin by nick-translation was used to analyze the FISH probe signal distribution in a different color. The probe signals proved to be a subset of the target signals remaining after denaturation and hybridization. We argue that the discontinuity of probe signals in Fiber-FISH is mainly caused by loss of target DNA and limited accessibility due to in situ renaturation and attachment. Furthermore, we conclude that FISH sensitivity is determined by hybridization efficiency and not the ability to generate sufficient signal from small probes. (J Histochem Cytochem 48:743-745, 2000)     

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Fluorochrome staining of multilamellar liposomes.

Multilamellar liposomes can be stained with such fluorochromes as acridine orange, eosin Y, neutral red, and thiazine red. The liposomes are brought into a 1% solution of the fluorochrome; 5-10 minutes later they are centrifuged and washed by resuspending in water or phosphate buffered saline three times. The last pellet is resuspended and a drop studied with the fluorescence microscope (1000 x magnification). The fluorochrome is seen to be accumulated in the liposomal membranes. Acridine orange could also be trapped in the aqueous compartments of the liposomes but the trapped fluorochrome was gradually lost from the liposomes. Part of the fluorochrome, however, remained associated with the liposomal membranes for a long time. Additional experiments justify the conclusion that an equilibrium is maintained between fluorochromes in the aqueous and lipid phases.     

Flavonoid-specific staining of Arabidopsis thaliana.

Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana.     

Improved staining boxes for fast, uniform staining of ultrathin sections on grids.

A standard LKB (LKB-Produkter Ab. S-161, 25 Brommma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. The staining boxes can be cut out to any required size or shape. The polymethyacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant than polymethyacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. For staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. The separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.     

Lectin staining of cultured CNS microglia.

Carbohydrate binding proteins, known as lectins, bind to specific sugar groups on most membranes. We used fluorescent and light microscopy to study the interaction of various lectins with the membranes of microglia cultured from neonatal rat or fetal mouse cerebral cortices. Microglia stained intensely with GS-1, RCA, WGA, and ConA and slightly with DBA, UEA, BPA, and SBA. No staining was seen with GS-2, MPA, or PNA. Staining was specific for microglia in the mixed glial cultures and was dose dependent. In addition, microglial lectin binding could be reduced or blocked by competitive inhibition using specific sugars. Treatment of the microglia with agents such as dimethylsulfoxide (DMSO), interleukin-1 (IL-1), interferon (IFN), or lipopolysaccharide (LPS) did not eliminate lectin staining, although the degree of staining was altered. Positive staining of the microglia was also associated with a functional change for at least one lectin, i.e., ConA. Superoxide anion production by microglia was increased in the presence of ConA. Overall, binding of the lectins GS-1, RCA, WGA, and ConA can be used as an identifying tool for microglia in glial cultures, but intensity of staining varies depending on their functional state.     

Dansylcadaverine specific staining for transamidating enzymes.

Versatile fluorescent staining methodologies, based on the incorporation of dansylcadaverine[N-(5-aminopentyl)-5-dimethylamino-l-naphthalenesulfonamide] into N,N-dimethylcasein, are described for the detection of transamidating enzymes of the endo-γ-glutamine:ε-lysine transferase type. Activity staining was employed for comparing the electrophoretic behaviors of such transamidating enzymes derived from human and guinea pig tissues. Two enzymatically active forms of guinea pig liver transglutaminase were found.     

Cobalt staining of Actinia equina nematocysts.

Nematocysts of Actinia equina are stained black by incubation in 2% CoCl2 followed by an aqueous wash and H2S treatment. They are also stained positively by morin. Nematocysts isolated from the acrorhage were found to have a high concentration of calcium of which only 30% was "free." It is suggested that the high concentration of calcium in the nematocysts accounts for their staining by cobalt and morin. Cobalt staining offers a simple and effective technique for investigation of nemotocysts.     

Silver carbonate staining reveals mitochondrial heterogeneity.

Silver staining methods, when selective, yield a high-contrast and high-resolution image in optical microscopy. A classical method for silver impregnation of mitochondria has been applied to murine tissues and reveals a marked heterogeneity among mitochondria in single cells. This heterogeneity can be detected in the optical microscope but is even more evident at the ultrastructural level. The differences in staining intensity may reflect different stages in the mitochondrial life cycle. The progressive accumulation of uranyl-argyrophilic material may be a marker of mitochondrial aging. This highly selective staining procedure may be of use in studies of mitochondrial changes under pathological conditions and during apoptosis.     

Aldehyde-fuchsin staining of antral endocrine cells.

Correlative studies have been made to identify the endocrine cells stained by Aldehyde-Fuchsin (AF) in the cat and human antral mucosa. D-cells weakly stained by AF after oxidation: this staining might be related to the disulphide group present in the molecule of Somatostatin (the hypothalamic hormone recently localized in D-cells). The gastrin-producing G-cells were on the contrary strongly stained (without oxidation) in tissues fixed in Helly-fluid. Histochemical investigations on the mechanism of such staining lead to the conclusion that it might be related to affinity of the stain for hidden acidic groups.     

Alternative staining methods for Lowicryl sections.

A number of stains and stain combinations have been identified that, when used with the hydrophilic resin Lowicryl K11M, produce marked improvements over aqueous uranyl and lead salts (UA-Pb) in terms of low granularity, specificity, and range of components contrasted. Three test specimens, tobacco mosaic virus (TMV), starfish sperm, and cultured mouse fibroblasts, were used to evaluate stain characteristics. UA-Pb showed a preference for nuclei acids, which were stained specifically by osmium ammine-B at pH 1.5. A number of stain combinations in which UA was followed or preceded by salts containing barium, manganese, tungsten, molybdenum, and vanadium provided excellent staining of protein-containing components, each stain combination being unique in terms of the degree to which specific components were discriminated. These stains were particularly effective for visualizing internal components of the nucleus where a number of fibrillar and particulate structures not seen with UA-Pb were well contrasted.     

Histochemical staining of lymphatic anchoring filaments.

Lymphatic anchoring filaments are stained by means of histochemical methods that demonstrate disulfide-groups. Thiosulfation of sections either followed by aldehyde-fuchsin staining or by Alcian Blue +0.8 M MgCl2 staining has been used. Lymphatic anchoring filaments display striking fine structural similarities to "elastic fiber microfibrils" and both kinds of fibers are characterized by disulfide content.     

A new staining technique for microfilariae.

A rapid whole mount staining method is described to identify and differentiate microfilariae without elaborate processing. A single solution combining Hoyer's mounting medium and hematoxylin stain facilitates light microscopic examination of nuclei and sheaths of microfilariae. The new technique stains microfilariae adequately in three to seven minutes at 60--64 C making the method preferable to conventional methods that may take as long as 45 to 60 minutes. Lantern heat may be used to heat slides in rural areas with good results.