Immunofluorescence Microscopy cell Biology Ptactical 3

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Presentation transcript:

Immunofluorescence Microscopy cell Biology Ptactical 3

Immunofluorescence Microscopy: When an antibody, or the antiimmunoglobulin antibody used to detect the antibody is labeled with a fluorescent dye This method is used when looking at the subcellular location of a protein of interest

Immunofluorescence Microscopy:

Technique: Common dyes: fluorescein, rhodamine Dyes chosen are excited by a certain light wavelength, usually blue or green, and emit light of a different wavelength in the visible spectrum Eg. Fluorescein emits green light Eg. Rhodamine emits orange/red light By using selective filters in a fluorescence microscope only the light from the dye is detected Available fluorescent labels now include red, blue, cyan or yellow fluorescent proteins

Uses: This can be used to detect the distribution of any protein By attaching different dyes to different antibodies the distribution of two or more molecules can be determined in the same cell or tissue sample

FLUORESCENCE MICROSCOPY

Types of immunofluorescence 1-Primary (or direct) 2-secondary (or indirect)

Direct Immunofluorescent

Indirect Immunofluorescent

Worksheet Practical 3 immunofluorescence Q1. What is the Principle of Fluorescence? Q2 What are the: Advantages of direct immunofluorescence ? Disadvantages of direct immunofluorescence ? Advantages of indirect immunofluorescence? Disadvantages of indirect immunofluorescence ? Q2. what are the Limitations of IF Techniques?