Immunofluorescence Technique Overview

Immunofluorescence

• Immunofluorescence is a technique used to visualize specific antigens.
• It involves binding a specific antibody, chemically conjugated with a fluorescent dye, to the antigen.

Definition

• Immunofluorescence is a technique that visualizes a specific antigen by binding a specific antibody conjugated with a fluorescent dye.
• Fluorochromes (e.g., fluorescein isothiocyanate (FITC), tetramethylrhodamine) absorb short UV wavelengths and emit longer wavelengths (visible light) such as green or red light.
• This technique aids in making Viral Plaques more visible.

Principle of Immunofluorescence (IF)

• IF is based on specific antigen-antibody interaction.
• A labeled antibody binds to its corresponding antigen.
• UV or specific wavelengths of light cause the fluorescent dye to emit visible light.
• This allows visualization of antigen-antibody complexes.

Principle of the Test (Diagram)

• Cells with specific human antigens are involved.
• Specific antibodies are labeled with a fluorescent dye like FITC.
• The labeled antibodies bind to the target antigens on the cells.
• Complexes of antigens and antibodies are visualized.

Requirements

• A slide
• Antibody tagged with a fluorescent dye.
• A fluorescent microscope.
• The specimen (e.g., tissue sections or cells).

Methods of IF Assay

• Two types of assays exist: direct and indirect.

Direct IF Assay (DIFA)

• A single fluorescent antibody binds directly to the target antigen.
• Sample preparation involves fixing tissues or cells to a slide.
• Applying the fluorescent antibody to the target antigen (if present).
• Looking at the slide under a fluorescence microscope.
• Fluorescence indicates a positive result (antigen presence).

Indirect IF Assay (IDIFA)

• Detects antibodies in a biological sample (e.g., serum).
• A slide/substrate is coated with a specific antigen.
• The patient serum sample is applied to the slide, with antibodies binding to the antigen (if present).
• A secondary antibody, labeled fluorescently, binds to the patient's antibodies.
• Observed with a fluorescence microscope, fluorescence indicates a positive result (antibody presence).

Result

• A confocal image showing phosphorylated AKT (green) in cardiomyocytes infected with adenovirus.

Comparison of Direct and Indirect IF