fluorescence Protocols

Category: Yeast Protocols: Yeast Nucleic Acid Protocols

Protocol for fluorescence in situ hybridization. - [Read Fluorescence in situ Hybridization Protocol]

Category: Molecular Imaging: Immunofluorescence Microscopy

Fluorescence Mounting Medium (Antifade). Spector Lab, CSHL. - [Read Fluorescence Mounting Medium (Antifade)]

Category: Immunology: Immunofluorescence Protocols

Protocol for fluorescence mounting medium (antifade). - [Read Fluorescence Mounting Medium (Antifade) Protocol]

Category: Immunology: Immunocytochemistry Protocols

Staining Cells in Suspension, Staining Cells Smeared on Slides. Immunofluorescent Technique. R & D Systems. - [Read Fluorescence Protocol Immunocytochemistry]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Immunofluorescence Staining Protocols

Fluorescent dyes absorb light at certain wavelengths and in turn emit their fluorescence energy at a higher wavelength. Each dye has a distinct emission spectrum, which can be exploited for multicolor analysis. eBioscience antibodies are available conjugated to a wide variety of fluorochromes. - [Read Fluorescent Dyes for Flow Cytometric Analysis]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Most biological specimens are relatively transparent, so details of internal and intracellular morphology are difficult to image in untreated living specimens using simple bright-field techniques. Fluorescence microscopy offers greater advantages and possibilities for increasing contrast and determining the specific localization of molecules in cells. Article outlines the three methods most commonly used to introduce an appropriate label into Drosophila tissue without perturbing the process. - [Read Fluorescent Reagents for Live Cell Imaging and Their Introduction into Cells]

Category: DNA Protocols: SNP Protocols

FP-TDI Method for SNP Detection. The FP-TDI protocol was originally reported by Drs. Chen, Levine, and Kwok at Washington University in 19991,2. FP-TDI stands for template directed dye terminator incorporation assay with detection by fluorescence polarization. It is a single base primer extension assay couple with homogeneous FP detection. Perkin Elmer - [Read FP-TDI Method for SNP Detection]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

Protocol describes an assay that relies on the endothelial cells’ ability to form distinct blood-vessel-like tubules in an extracellular matrix  where they can subsequently be visualized by fluorescence microscopy.  Although quantification of the tubules can be performed by manual tracing, this method precludes the use of the assay in unbiased high-throughput applications. - [Read Image Based Assay of Endothelial Cell Tube Formation Protocol]

Category: Microinjection Protocols

Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. Includes: Pretreatment of metaphase spreads on slides; Denaturation and hybridization; Single color fluorescent detection with immunological amplification; Multicolor fluorescence in situ hybridization (Multicolor FISH); Results obtained with human metaphase chromosome spreads. - [Read In Situ Hybridization to Human Metaphase Chromosomes using DIG-, Biotin- or Fluorochrome-Labeled DNA]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Article presents an introduction to fluorescence microscopy. Includes: Fundamentals of Excitation and Emission; Stokes' Shift; Fading, Quenching, and Photobleaching; Fluorescence Light Sources; Filter Terminology; The Fluorescence Light Budget; Detecting Single Molecules. - [Read Introduction to Fluorescence Microscopy]

Category: Molecular Imaging: Light Microscopy

Light Microscopy - Microscopes in Cell Biology. House Ear Institute. Fluorescence microscopy, Nomarski differential interference contrast, Comparison between phase contrast and interference contrast optical systems , Interference contrast, Phase contrast, Darkfield illumination, alignment of Kohler illumination system, Protocol for using oil immersion lenses, Use of immersion oil, Calculating the final magnification on the photomicrograph, vibration, The coverslip glass, Photomicroscopy. - [Read Light Microscopy - Microscopes in Cell Biology]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope when three planes along the z-axis of the cell are acquired. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP & Bright Field—Three Z Axis]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Image Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP and Bright Field Images]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent images of live cells expressing yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. Protocol includes: Description of Microscope and Imaging Setup; Description of Acquisition Parameters; Movie Processing. - [Read Live Cell Spinning Disk Confocal Fluorescence Imaging of Cells- YFP Time Series for Markers]

Category: Immunology: B Cell Protocols

This assay is used to measure cell viability. It is a two-color fluorescence assay that simultaneously determines: Live cell number and Dead cell number. - [Read Live Dead Assay for Cell Viability Protocol]

Category: C. elegans Protocols

At low magnification, dissection microscopes are useful, whereas at higher magnification and resolution, a wide range of optical methods can be used, including differential interference contrast (DIC) optics, phase contrast, fluorescence, polarized light, confocal, and dark field. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method to assess concentrations of free cytoplasmic calcium, [Ca2+]i, for cultured adherent RAW 264.7 cells in an 8-well coverglass. This objective is accomplished using the Ca2+-sensitive fluorescent dye, fura-2 acetoxymethyl (AM), which permeates cell membranes as an ester and is hydrolyzed in the cell to its Ca2+-sensitive acidic form. Fluorescence for the adherent cells is measured over time with cells that have been washed free of extracellular dye. - [Read Live Single-Cell Fura-2 Measurements to Determine the Intracellular Free Calcium]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Assay measures cell viability. It is a two-color fluorescence assay that simultaneously determines Live cell number and Dead cell number. This protocol is designed for use with the GEMINI XS Microplate Spectrofluorometer, a multi-well plate scanner with dual excitation/emission capabilities, but the assay is also adaptable for flow cytometry and fluorescence microscopy. Includes: Cell Culture; Preparation for the Assay; Live/Dead Assay; Reading the Plate; Data Analysis; Alternative protocol. - [Read Live/Dead Assay for Cell Viability Protoco]

Category: Molecular Imaging

The Handbook — A Guide to Fluorescent Probes and Labeling Technologies is a comprehensive resource for fluorescence technology and its applications. Newly revised, The Handbook contains detailed information describing the use of more than 3000 Molecular - [Read Molecular Probes Handbook Invitrogen]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Multiphoton fluorescence microscopy is a powerful new technology that enables the acquisition of optical sections without the use of a pinhole aperture typically used for confocal microscopy. The technique is based upon the two-photon principle: A fluorescent molecule simultaneously absorbs two photons producing an electronic transition from the ground to excited state equal to two times the energy of each incident photon. - [Read Multiphoton Images from LSM 510 NLO System]

Category: Molecular Imaging

Order Fluorophore Wall Chart. Free. CellScience - [Read Order Fluorophore Wall Chart]

Category: Cytoskeleton Protocols: Intermediate Filaments Protocols

Intermediate filaments (IF) are major cytoskeletal systems of vertebrate and many nonvertebrate cells whose expression is cell-type specific and developmentally regulated. This protocol describes the x-rhodamine labeling of one type of IF, vimentin, and a method for microinjection of the labeled vimentin into cultured cells. IF dynamics can then be examined with fluorescence microscopy. - [Read Preparation and Microinjection of x-Rhodamine-Labeled Vimentin Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

Category: Cytogenetics Protocols

An ideal method of tissue preparation ensures both good specimen morphology and that the target molecules are in the optimum state for probe access and hybridization. DNA:DNA in situ hybridization is usually carried out on chromosome spread preparations where chromosome and nuclei are released from cells and spread on a glass microscope slide. This method yields well separated and enlarged chromosomes with good morphology which can be analyzed in transmitted light or fluorescence microscopes. - [Read Preparation of Chromosome Spreads]