Immunofluorescence Assay Test

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:

1. Direct Immunofluorescence: Only one antibody is used. It’s called the primary antibody. It is tagged with a fluorescent stain.
2. 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.