Fluorescence and Confocal Microscopy Outline

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.

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.