Immunofluorescence Microscopy - 3. Assay Formats for Immunofluorescence Microscopy

There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect).

1. Primary (direct)

   
   Primary, or direct, immunofluorescence uses a single antibody that is conjugated directly to a fluorescent dye. The antibody recognizes the target molecule, binds to it and the conjugated fluorescent dye can be detected by the microscope. This technique, which reduces the number of steps in the staining procedure and is therefore faster, can avoid issues with antibody cross-reactivity or non-specificity which can lead to increased background signal. However, this method lacks any signal amplification inherent for the indirect method and requires laborious efforts by the scientist to conjugate potential numerous different primary antibodies required.




2. Secondary (indirect)

   
   Secondary, or indirect, immunofluorescence uses two antibodies; a primary antibody which recognizes the target biomolecule and binds to it and a secondary antibody conjugated to a fluorescent dye, which recognizes and binds to the primary antibody and indirectly localizes the target for detection by the microscope. While this protocol is more complex than the direct method, it is more flexible with regard to experimental design, results in greater signal detection through amplification and is easier in that secondary antibody conjugates are commercially available.

Immunofluorescence Microscopy

1. Background
2. Types of Fluorescent Microscopes
3. Assay Formats for Immunofluorescence Microscopy
4. List of Fluorescent Labels Used for Antibody Conjugation by Rockland
5. ATTO-TEC Fluorescent Dye Conjugates
6. CyDye® Fluorescent Dye Conjugates
7. Dylight™ Fluorescent Dye Conjugates
8. IRDye® Fluorescent Dye Conjugates
9. Other Fluorescent Dye Conjugates