A Conjugate of Cellulase with Fluorescein Isothiocyanate: A Specific Stain for Cellulose

A fluorescent technique has been developed for in situ staining of cellulose. The staining agent is a conjugate of cellulase and fluorescein isothiocyanatc (FITC). Application of this agent does not disturb intercellular or intracellular substances. The technique depends on the specific binding of the fluorescent labeled enzyme to its substrate. The stain has been tested on cell-free noncellulose polysaccharides similar to cellulose and does not stain them. The technique has been used to localize cellulose during the life cycle of Dictyostelium discoideum with results that correspond to previous work using other methods.     

EVIDENCE FOR GLUCAN COMPONENTS IN THE PRIMARY ZYGOTE WALL OF CHLAMYDOMONAS MONOICA

The primary zygote wall of C. monoica is transient and is released from mature zygospores. The fluorochromes aniline blue and primulin, used in other systems to detect β-1,3 glucans, stain the primary wall intensely. Two β-1,3 glucan synthases have been identified in higher plants: a calcium-dependent synthase produced in response to wounding and induced by chitosan, and a magnesium-dependent enzyme, associated with pollen development and unresponsive to chitosan. Chitosan has no effect on C. monoica primary wall synthesis or staining properties. We are presently testing for the effect of magnesium and/or calcium depletion on primary wall synthesis. Aniline blue and primulin do not stain purified cellulose fibers, while the fluorochrome Calcofluor does. Calcofluor also stains the primary wall intensely. For all fluorochormes tested, fluorescence is first detected in motile quadriflagellate zygotes. Aniline blue staining maximizes quickly, while Calcofluor staining continues to intensify until primary wall release. Dinitrobenzonitrile, a specific inhibitor of cellulose synthesis in plants, has no effect on primary wall synthesis in C. monoica. Addition of glucanase or cellulase to partially purified primary walls results in wall thinning and loss of staining. Using electron microscopy, we are evaluating the effects of these enzymes on primary wall ultrastructure. Further studies are needed to determine whether all three fluorochromes are recognizing the same polysaccharide component (a β-1,3 glucan or a β-1,3; β-1,4 mixed glucan), or whether Calcofluor staining indicates the presence of a distinct component containing β-1,4 linkages, such as cellulose or a xyloglucan.     

An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies

Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.     

Use of a Fluorescent Brightener to Demonstrate Cellulose in the Cellular Slime Molds

The presence and location of cellulose in different stages of the life cycles of the cellular slime molds can be demonstrated by use of the disodium salt of 4,4'-bis(4-anilino-6-bis (2-hydroxyethyl)-amino-s-triazin-2-ylamino)-2,2' -stilbene disulfonic acid, a fluorescent brightener. It may be used successfully as a direct stain at a concentration of 0.1% in half-normal saline at pH 6; and it may be incorporated into growth media as a vital stain at a concentration of 0.0025% with no inhibitory effect at any developmental stage. Vegetative myxamoebae contain no cellulose and show no fluorescence in the presence of this brightener when viewed with ultraviolet light. In later stages of the life cycle, the time and sites of cellulose formation can be demonstrated with the brightener because of its fluorescence. e.g., in the slime covering of the pseudoplasmodia, in the sorophore sheath, in the walls of stalk cells and spores, in the walls of microcysts, and in the walls and sheath material of macrocysts. The brightener appears to be a very sensitive indicator for cellulose, and it has certain advantages over other cellulose stains, since the staining reaction (fluorescence) is very intense, long-lasting, and not obscured by unstained cellulose-free myxamoebae if such are present.     

A highly fluorescent simultaneous azo dye technique for demonstration of nonspecific alkaline phosphatase activity.

We describe a fluorescent histochemical technique for detection of nonspecific alkaline phosphatase (APase) in cells. The technique utilizes standard azo dye chemistry with naphthol AS-MX phosphate as substrate and fast red TR as the diazonium salt. The reaction product is a highly fluorescent red precipitate. Pre-implantation mouse embryos were used to establish optimal fixation and staining protocols and the specificity and sensitivity of the method. Fixation was in 4% paraformaldehyde for 1 hr, as glutaraldehyde induced autofluorescence of the cells. Maximal discriminable staining was detected after 15-20 min in the stain solution. The stain solution itself proved to be non-fluorescent, thus allowing visual observation of the progress of the staining reaction by fluorescence microscopy in its presence. To test the specificity of this fluorescent APase stain, a variety of cell types of known APase reactivity were stained by this protocol. Mouse lymphocytes and STO fibroblasts were negative, whereas F9 teratocarcinoma cells, intestinal epithelial cells, and rat fetal primordial germ cells were all found to be highly positive for APase activity, in agreement with published results on APase localization in these cells.     

7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry

A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.     

A RAPID SIMPLE TECHNIQUE UTILIZING CALCOFLUOR WHITE M2R FOR THE VISUALIZATION OF DINOFLAGELLATE THECAL PLATES1

The fluorescent stain Calcofluor White M2R readily binds to cellulose and other β-linked glucans (Hughs and McCully 1975). We have found the stain to readily bind to the thecal plates of armored dinoflagellates. Considerable detail is revealed about plate structure in both living and preserved specimens at the light microscopic level. This simple rapid technique should prove useful for the tabulation and study of dinoflagellate thecae.     

Ultrafast coelectrophoretic fluorescent staining of proteins with carbocyanines

Protein detection on SDS gels or on 2-D gels must combine several features, such as sensitivity, homogeneity from one protein to another, speed, low cost, and user-friendliness. For some applications, it is also interesting to have a nonfixing stain, so that proteins can be mobilized from the gel for further use (electroelution, blotting). We show here that coelectrophoretic staining by fluorophores of the oxacarbocyanine family, and especially diheptyloxacarbocyanine, offers several positive features. The sensitivity is intermediate between the one of colloidal CBB and the one of fluorescent ruthenium complexes. Detection is achieved within 1 h after the end of the electrophoretic process and does not use any fixing or toxic agent. The fluorescent SDS-carbocyanine-protein complexes can be detected either with a laser scanner with an excitation wavelength of 488 nm or with a UV table operating at 302 nm. Excellent sequence coverage in subsequent MS analysis of proteolytic peptides is also achieved with this detection method.     

A fluorescent antibody staining technique to detect bacterial adherence to urinary tract epithelial cells

A fluorescent antibody technique has been devised to assess specifically the adherence of Escherichia coli in vitro to uroepithelial cells from healthy women and bacterial adherence in vivo to cells from women with symptomatic urinary tract infection. Similar values can be obtained using methylene blue as the bacterial stain, but this depends on the experience of the observer. The results indicate that E. coli adherence to uroepithelial cells is a factor in the infection process. We suggest that uroepithelial cells from patients with symptoms of a urinary tract infection whose urine has a low bacterial count (less than 10(3) cells/ml) could be examined for the presence of adherent uropathogens, which may be indicative of an infection. Although the fluorescent staining technique possibly would be expensive, the results would be specific and reliable. Other diagnostic and research applications suggest themselves as in studies of bacterial colonization of mucosal tissues or plastic catheters, where conventional light microscopy and radiolabelling methods are not effective.     

Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.     

The use of an optical brightener in the study of plant structure.

An optical brightener Calcoflour White M2R New has been used to stain cell walls of higher plants. It can be used either as a vital stain for intact plants or for hand sections and plastic-embedded thin sections. Walls are brilliantly fluorescent while most cytoplasmic components are normally unstained. The brightener binds strongly to cellulose, carboxylated polysaccharides, and callose. Staining for 20 sec to 2 min in a 0.01% solution of the brightener is preferred for most purposes.     

Silver stain for proteins on a cellulose acetate membrane

A rapid and sensitive silver staining method to detect proteins on a cellulose acetate membrane has been established. This method is achieved by modification of the silver-based color staining for detection of proteins in polyacrylamide gels [D. W. Sammons, L. D. Adams, and E. E. Nishizawa, Electrophoresis 2, 135-141 (1981)] and applied to our new type of two-dimensional electrophoresis for analysis of proteins on a cellulose acetate sheet [T. Toda, T. Fujita, and M. Ohashi, Anal. Biochem. 119, 167-176 (1982)]. Maximal sensitivity of silver stain for proteins on a cellulose acetate membrane can be obtained by an optimal balance between deposition of silver on the protein and on the background. Certain kinds of proteins are colored red, orange, or grayish-blue. The silver stain is 20-80 times more sensitive than Coomassie blue and some spots are visualized reproducibly by silver only. Densitometric evaluation of standard proteins stained with silver and Coomassie blue is also demonstrated. The method takes only 50 min to perform and is sensitive, simple, and reproducible.     

Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations

A new fluorescent stain has been developed for detecting cloned beta-galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry. The staining reaction is based on enzymatic cleavage of alpha-naphthol-beta-D-galactopyranoside by intracellular beta-galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product. This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible beta-galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication. The method enables rapid determination of the fraction of plasmid-containing cells as well as quantitation of intracellular beta-galactosidase content by kinetic enzyme assay. Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.     

Triplet quantum yields in light-scattering powder samples measured by laser-induced optoacoustic spectroscopy (LIOAS)

In recent years, different methods and techniques have been applied to study the primary photophysical processes occurring in dye-loaded light-scattering powdered samples. In spite of this, there are still no reliable methods for the determination of triplet quantum yields for this kind of systems. Laser-induced optoacoustic spectroscopy (LIOAS) has been extensively used for the determination of triplet quantum yields of dyes in solution. In a previous work, LIOAS was applied to the measurement of absolute emission quantum yields of highly fluorescent powdered samples. Excellent agreement was found with values obtained from reflectance data. In this work, we apply the same technique for the determination of triplet quantum yields of Rose Bengal and Erythrosine B adsorbed on microcrystalline cellulose. In contrast to water and other solvents, internal conversion cannot be neglected in the cellulose environment. The triplet quantum yield for both dyes is around 0.55 and does not change with dye concentration.     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

Alcian blue as a protein stain for sodium alkyl sulfate-polyacrylamide gels: Application to analysis of polypeptide components of the human platelet

The cationic dye, Alcian blue, previously used as a glycoprotein-specific stain on cellulose acetate and polyacrylamide gels, was found to be capable of staining a variety of purified proteins and each of the components of the human platelet presently identifiable with Coomassie blue R or periodic acid-Schiff (PAS) reagent in sodium alkyl sulfate-polyacrylamide gel electrophoretic preparations. Evidence was obtained to indicate that staining of detergent-protein complexes by Alcian blue occurs by virtue of the affinity of the stain for accessible sulfate groups of detergent molecules, especially sodium tetradecyl sulfate, hydrophobically associated with polypeptide chains. Thus, Alcian blue fails to stain nonglycosylated proteins when pure sodium dodecyl sulfate (C₁₂) is used as the detergent, but does so readily when small quantities of sodium tetradecyl sulfate are also present. The advantages of using Alcian blue to determine platelet protein composition and to make quantitative comparisons between bands in sodium alkyl sulfate gels are discussed.     

Zinc-Specific N-(6-Methoxy-8-Quinolyl)-Para-Toluenesulfonamide as a Selective Nontoxic Fluorescence Stain for Pancreatic Islets

Separation of the endocrine from the exocrine pancreatic tissue by fluorescence activated sorting has been limited by the lack of an ideal fluorescent label for islet tissue. Our studies indicates the zinc-specific stain N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), has characteristics ideal for use as a fluorescent label for islet tissue. Dispersed rat pancreas cells stained with TSQ produced bright blue fluorescence when excited by UV light [peak emission wavelength at 480 nm. maximal excitation at 365 nm). The fluorescence was specific for islet tissue as confirmed by counterstaining with the islet-specific stain dithizone and there was minimal background staining of exocrine tissue. Stained tissue remained brightly fluorescent for 2 hr. with some fading by 4 hr. Injection of TSQ into rats at a concentration sufficient to produce staining of islets produced no toxicity discernible at 4 months. The viability of isolated rat islets stained with TSQ was maintained as shown by supravital staining, in vitro secretion of insulin, and reversal of diabetes after transplantation of stained islets into diabetic syngeneic recipients.     

A new, rapid, microwave-stimulated method of staining melanocytic lesions

A new and sensitive method of staining melanocytic lesions is described. Tissue sections covered by a solution of colloidal silver nitrate are exposed to microwaves for 45 sec in a domestic oven to produce clean, crisp staining of melanocytes and melanoma cells, often showing long delicate dendritic cell processes. The staining technique does not stain other pigments or argyrophilic tissues and is shown to be more sensitive than the standard Masson-Fontana procedure.     

A New, Rapid, Microwave-Stimulated Method of Staining Melanocytic Lesions

A new and sensitive method of staining melanocyte lesions is described. Tissue sections covered by a solution of colloidal silver nitrate are exposed to microwaves for 45 sec in a domestic oven to produce clean, crisp staining of melanocytes and melanoma cells, often showing long delicate dendritic cell processes. The staining technique does not stain other pigments or argyrophilic tissues and is shown to be more sensitive than the standard Masson-Fontana procedure.     

A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.