In Situ Immunofluorescence Analysis: Immunofluorescence Microscopy
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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.
Fig. 1. In situ immunofluorescence detection of transcription factors at intranuclear sites. Runx/Cbfa/AML transcription factors provide an example of regulatory proteins that can be detected in situ . HeLa cells grown on gelatin-coated cover slips were transiently transfected with 0.5 μg of Runx2 expression plasmid, using “SuperFect” reagent (Qiagen Inc, CA). Cells were processed 20 h later for in situ detection of Run μ2 in intact cells (A) or after removal of cytoskeletal component (B) or in nuclear matrix preparations (C) . Run μ2 proteins were detected with a rabbit polyclonal Run μ2 antibody and an fluorescein isothiocyanate-conjugated antirabbit secondary antibody. DAPI detects deoxyribonucleic acid (DNA) in nuclei of whole cells and CSK extracted cells but not in NMIF preparations because DNA has been digested and extracted. Differential interference contrast microscopy shows a bright field image of cells. The punctate, non-nucleolar distribution of Run μ2 protein is preserved throughout the extraction procedure. Original magnification � 63.
