Fluorescence and Confocal Microscopy Outline
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Questions and Answers

What type of chemicals are fluorochromes?

  • Proteins
  • Enzymes
  • Photoreactive chemicals (correct)
  • Antibodies

    • What happens to the light absorbed by fluorochromes?

  • It is reflected back as white light
  • It is converted into heat energy
  • It is absorbed completely by the fluorochromes
  • It is emitted at a lower energy level as fluorescence (correct)

    • What is the primary function of the secondary antibody conjugated to a fluorochrome in immunofluorescent staining?

  • To allow detection of antigen-antibody complexes (correct)
  • To bind to the antigen
  • To emit high energy light
  • To locate and identify patterns of protein expression

    • What type of tissues is immunofluorescent staining suitable for?

    <p>Frozen, non-fixed tissues and ethanol fixed tissues</p> Signup and view all the answers

    What is the main purpose of immunofluorescent staining?

    <p>To identify patterns of protein expression in cells</p> Signup and view all the answers

    What is the role of fluorophores in fluorescence microscopy?

    <p>To absorb light and emit it with lower energy</p> Signup and view all the answers

    What is the purpose of incubating cells with a blocking solution in the immunofluorescent cell staining process?

    <p>To minimize non-specific staining</p> Signup and view all the answers

    Which fluorochrome is the secondary antibody directed against the primary antibody in the provided information?

    <p>FITC</p> Signup and view all the answers

    What is used to visualize stained specimens on a fluorescent microscope?

    <p>PBS/Glycerol mix</p> Signup and view all the answers

    What is the major problem in microscopic examination of fluorescent specimens?

    <p>Photobleaching</p> Signup and view all the answers

    Which chemical agent may be added to the mounting medium to preserve specimen brightness and prevent photobleaching?

    <p>Trans-pyridine-2-azo-p-dimethylaniline (PADA)</p> Signup and view all the answers

    What is the purpose of incubating fixed cells in 1% Triton X-100 in PBS+0.02%BSA for 2 minutes at room temperature?

    <p>To permeabilize the cells</p> Signup and view all the answers

    Which primary antibody is specific for the protein p53 as mentioned in the provided information?

    <p>Sheep anti-p53 polyconal</p> Signup and view all the answers

    What is the primary purpose of immunofluorescent staining?

    <p>To locate and identify patterns of protein expression in cells.</p> Signup and view all the answers

    What is the function of fluorochromes in fluorescence microscopy?

    <p>Fluorochromes absorb light in a specific wavelength range and re-emit it with lower energy, allowing the visualization of fluorescent samples.</p> Signup and view all the answers

    What type of tissues is immunofluorescent staining suitable for?

    <p>Frozen, non-fixed tissues and ethanol fixed tissues.</p> Signup and view all the answers

    What happens to the light absorbed by fluorochromes?

    <p>Fluorochromes re-emit the absorbed light at a longer wavelength with lower energy, a process known as fluorescence.</p> Signup and view all the answers

    What is the major advantage of confocal microscopy over conventional fluorescent microscopy?

    <p>Confocal microscopy provides higher resolution and better 3-dimensional imaging compared to conventional fluorescent microscopy.</p> Signup and view all the answers

    What is the role of the secondary antibody conjugated to a fluorochrome in immunofluorescent staining?

    <p>To bind to the antibody-antigen complex and allow detection of the complex through fluorescence.</p> Signup and view all the answers

    What chemical can be added to the mounting medium to preserve specimen brightness and prevent photobleaching?

    <p>trans-pyridine-2-azo-p-dimethylaniline (PADA)</p> Signup and view all the answers

    What is the specific fluorochrome used for the secondary antibody directed against the primary antibody in the provided information?

    <p>FITC</p> Signup and view all the answers

    In immunofluorescent cell staining, what is the primary function of the secondary antibody conjugated to a fluorochrome?

    <p>To bind to the primary antibody and aid in visualization under a microscope.</p> Signup and view all the answers

    What is the role of fluorochromes in fluorescence microscopy?

    <p>Fluorochromes absorb light at one wavelength and emit it at a longer wavelength, producing fluorescence, which aids in visualization under the microscope.</p> Signup and view all the answers

    What chemical is used to fix cells during the cell preparation process?

    <p>Paraformaldehyde or methanol/acetone</p> Signup and view all the answers

    What is the major problem in microscopic examination of fluorescent specimens?

    <p>The tendency of fluorochromes to lose fluorescence upon excitation by a high energy light source, known as photobleaching.</p> Signup and view all the answers

    What is the purpose of incubating fixed cells in 1% Triton X-100 in PBS+0.02%BSA for 2 minutes at room temperature?

    <p>To permeabilize the cells, allowing antibodies to enter and bind to intracellular structures or proteins.</p> Signup and view all the answers

    What is used to visualize stained specimens on a fluorescent microscope?

    <p>Mounting medium, usually a PBS/Glycerol mix</p> Signup and view all the answers

    What is the specific primary antibody mentioned for the protein p53 in the provided information?

    <p>Sheep anti-p53 polyconal</p> Signup and view all the answers

    What is the specific fluorochrome used for the secondary antibody directed against the protein a tubulin in the provided information?

    <p>Texas Red</p> Signup and view all the answers

    Study Notes

    Fluorochromes & Immunofluorescent Staining

    • Fluorochromes are chemicals that absorb light at one wavelength and emit light at a longer wavelength.
    • Light absorbed by fluorochromes is re-emitted as fluorescence at a longer wavelength.
    • Secondary antibodies conjugated to a fluorochrome in immunofluorescence staining bind to the primary antibody, enhancing the signal and allowing for visualization.
    • Immunofluorescent staining is suitable for diverse tissues, including cells, tissues, and organisms.
    • The main purpose of immunofluorescent staining is to visualize specific molecules within cells or tissues.
    • Fluorophores are used in fluorescence microscopy to label specific targets within cells or tissues, allowing for their visualization.

    Immunofluorescent Cell Staining Process

    • Incubating cells with a blocking solution reduces non-specific binding of antibodies to the sample.
    • In the provided context, the secondary antibody is directed against the primary antibody using Alexa Fluor 488, a fluorochrome.
    • Fluorescent microscopes are used to visualize stained specimens.
    • The major problem in fluorescent microscopy is photobleaching, where the fluorescence signal fades over time.
    • Antifade mounting medium can be used to preserve specimen brightness and prevent photobleaching.
    • Incubating fixed cells in 1% Triton X-100 in PBS+0.02% BSA for 2 minutes at room temperature permeabilizes the cell membrane, allowing antibodies to access intracellular antigens.
    • The primary antibody specific for the protein p53 is anti-p53.

    Key Applications & Functions

    • The primary purpose of immunofluorescent staining is to localize and visualize specific target molecules in cells or tissues.
    • Fluorochromes act as fluorescent labels in fluorescence microscopy, allowing for the detection and visualization of specific targets.
    • Immunofluorescent staining is suitable for various tissue types, including cells, frozen sections, and tissues.
    • Confocal microscopy provides higher resolution images compared to conventional fluorescent microscopy, enhancing the level of detail observed.

    Chemical Components & Techniques

    • Formaldehyde is commonly used to fix cells for immunofluorescence staining.
    • Photobleaching is a major problem during fluorescent microscopy.
    • Incubating fixed cells in 1% Triton X-100 in PBS+0.02%BSA for 2 minutes at room temperature permeabilizes the cell membrane.
    • Fluorescence microscopes are used to visualize stained specimens.
    • The specific primary antibody mentioned for the protein p53 is anti-p53.
    • The specific fluorochrome used for the secondary antibody directed against the protein a tubulin is Alexa Fluor 568.

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    Quiz Team

    Description

    Learn about immunofluorescent staining, conventional fluorescent microscopy, confocal microscopy, and their applications in this presentation by Dr. Firas Zakaria and Dr. Hassan Khachfe. Understand the use of fluorochromes as fluorescent markers in microscopy and the specific requirements for suitable samples.

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