In Situ Immunofluorescence Analysis

Abstract

Immunofluorescence is one of the most widely used techniques to study the localization of transcription factors, proteins, and structural components of nuclear architecture and cytoarchitecture. High-resolution in situ immunofluorescence approaches permit assessment of functional interrelationships between nuclear structure and gene expression that are linked to the intranuclear compartmentalization of nucleic acids and regulatory proteins (an example is shown in Fig. 1). The success of this method is dependent on the quality and specificity of the antibodies and the relative stability of antigens. Generally, the overall scheme for localization of cellular proteins involves fixation and permeabilization of cells for antibody accessibility, blocking, and staining with specific antibodies before microscopic examination. To reveal the subcellular and subnuclear macromolecular complexes that comprise and govern activation of the regulatory machinery for gene expression, cells can be subjected to selective extractions before immunodetection as described below.