General Aim of Immunofluorescence Assay Test
To fluorescently label a target antigen in a tissue, and visualize it with a fluorescent microscope.
Method
Indirect Immunofluorescence Assay
Learning Objectives (ILOs) of Immunofluorescence Assay Test
To understand the basic concepts of the Immunofluorescence Assay.
To apply indirect immunofluorescence assay test protocol.
To learn how to perform cell fixation.
To practice the ‘washing technique’ skill.
Immunofluorescence Assay: Theoretical Background
- Immunofluorescence is an immunological technique designed to visualize antigens in cellular contexts.
- This is done by using antigens specific fluorescently tagged antibodies.
There are two types of Immunofluorescence techniques:
- Direct Immunofluorescence: Only one antibody is used. It’s called the primary antibody. It is tagged with a fluorescent stain.
- Indirect Immunofluorescence: Two antibodies are used. The secondary antibody is the one that is fluorescently stained.
Principle of Work
- We use the indirect immunofluorescence staining technique to detect an antigen inside cultured cells.
- To prepare cells, fixation and cell permeabilization are done.
- Afterward, a blocking buffer is added prior to the addition of the primary antibody, which is specific to the antigen to be detected.
- Washing steps are applied to get rid of the excess unbound antibodies.
- A secondary antibody is then added to label the antigen fluorescently so that it can be visualized on a fluorescent microscope.
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