Enhancement of Staining Intensity in the Fluorescent-Antibody Reaction

Pital, Abe; Janowitz, Sheldon L.

doi:10.1128/jb.86.4.888-889.1963

Cited by 23 publications

(6 citation statements)

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“…Attempts to enhance staining by increasing pH. Pital and Janowitz (1963) reported increased intensity of staining of bacterial smears when stained preparations were washed and mounted in 0.5 M carbonate buffer (pH 9.0). Attempts to do this with stained mumps and NDV cover-slip preparations were unsuccessful.…”

Section: Resultsmentioning

confidence: 99%

Preparation of Fluorescein Isothiocyanate-Labeled γ-Globulin by Dialysis, Gel Filtration, and Ion-Exchange Chromatography in Combination

1965

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DEDMON, ROBERT E. (Presbyterian-St. Luke's Hospital, Chicago, Ill.), ALBERT W. HOLMES, AND FRIEDRICH DEINHARDT. Preparation of fluorescein isothiocyanate-labeled 7-globulin by dialysis, gel filtration, and ion-exchange chromatography in combination. J. Bacteriol. 89:734-739. 1965.-Antiviral immune 7y-globulins isolated from rabbit and guinea pig sera were labeled through dialysis membranes with fluorescein isothiocyanate and purified in several ways to eliminate nonspecific staining. Gel filtration of the conjugate with Sephadex G-25 coarse beads followed by column fractionation with diethylaminoethyl-Sephadex yielded consistently highly specific staining materials. Fluorescein-protein ratios varied between 1.0 and 4.0. This technique has proved to be simple and reliable, and is less time-consuming than previous techniques.

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Section: Resultsmentioning

confidence: 99%

Preparation of Fluorescein Isothiocyanate-Labeled γ-Globulin by Dialysis, Gel Filtration, and Ion-Exchange Chromatography in Combination

1965

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“…Smears were stained directly with a drop of VEE fluorescein-labeled antibody solution for 30 min in a moist chamber at room temperature. After this, the slides were rinsed and washed for 10 min in carbonate-bicarbonate buffer (0.5 M, pH 9.0) and mounted in glycerol adjusted to pH 9.0 with carbonate-bicarbonate buffer (25). Labeled globulin was used undiluted and diluted 1:2 with neutral buffered saline.…”

Section: Methodsmentioning

confidence: 99%

“…Labeled globulin was used undiluted and diluted 1:2 with neutral buffered saline. Specifications for the fluorescence lamp and microscope used have been described previously (25). Slides were examined for specific staining of viral particles, and fluorescent reactions were graded on the basis of lowest to highest estimated intensity (1 + to 4+).…”

Section: Methodsmentioning

confidence: 99%

Hemagglutination-Inhibition Method and Immunofluorescence Staining with Venezuelan Equine Encephalomyelitis Virus

Shepel¹

1966

Applied Microbiology

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SHEPEL, MICHAEL (Fort Detrick, Frederick, Md.). Hemagglutination-inhibition method and immunofluorescence staining with Venezuelan equine encephalomyelitis virus. Appl. Microbiol. 14:346-352. 1966.-Hemagglutination and fluorescent antibody (FA) are compared for the direct detection of virus devoid of host cells. A determination was made of the minimal number of tissue plaque-forming units of Venezuelan equine encephalomyelitis virus that could be detected by the hemagglutination technique. Similar concentrations of the virus in bovine albumin borate saline, Brain Heart Infusion broth (Difco), and demineralized water were tested by the FA technique. Somewhat higher concentrations of the virus in bovine albumin borate saline were used in the hemagglutination-inhibition test. The quantitative hemagglutination procedure employed for these studies was carried out at 37 C for 75 min with variations in concentration of goose red cells. As a result of lowering the red cell concentration, smaller concentrations of virus were detected. The direct FA staining procedure applied to slide preparations containing known numbers of tissue culture plaque-forming units of virus was negative. Adsorbed viral antigen on agglutinated goose erythrocytes was visualized by direct and indirect FA techniques.

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“…Smears were covered with 0.1 ml of labeled enzyme and allowed to stain for 5 min in a moist chamber at 37 C. After staining, the slides were gently rinsed for 10 min in CBB, and smears were immediately mounted in glycerol adjusted to pH 9.6 with 2% CBB. Stained preparations were examined by fluorescence microscopy as described in a previous publication (21).…”

Section: Downloaded Frommentioning

confidence: 99%

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

Pital¹,

Janowitz²,

Hudak³

et al. 1967

Applied Microbiology

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Fluorescein isothiocyanate-labeled,-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gramnegative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed.

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Enhancement of Staining Intensity in the Fluorescent-Antibody Reaction

Cited by 23 publications

References 0 publications

Preparation of Fluorescein Isothiocyanate-Labeled γ-Globulin by Dialysis, Gel Filtration, and Ion-Exchange Chromatography in Combination

Preparation of Fluorescein Isothiocyanate-Labeled γ-Globulin by Dialysis, Gel Filtration, and Ion-Exchange Chromatography in Combination

Hemagglutination-Inhibition Method and Immunofluorescence Staining with Venezuelan Equine Encephalomyelitis Virus

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

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