Histology Protocols pp 43-57
Immunofluorescence Detection of the Cytoskeleton and Extracellular Matrix in Tissue and Cultured Cells
“A picture is worth a thousand words” goes the proverb. A poor picture however can be worse than saying nothing at all. This is particularly true for immunofluorescence pictures that in addition to their informative character bear an esthetic component. We here provide a panel of straightforward methods to process tissue sections and cultured cells for immunostaining of cytoskeletal elements, primarily those associated with actin filaments. We want to emphasize to the reader the fact that the choice of the processing method will have an important influence on the outcome of the immunostaining and thus on the interpretation of the results. Fixation of cultured cells with cross-linking reagents such as paraformaldehyde efficiently preserves structural elements at the expense of reduced antigenicity. The degree and timing of cell permeabilization with detergents, along with chemical cross-linking, contributes to the clarity and resolution of distinct structures but can also lead to loss of information. Fixation with organic solvents like methanol will, in most cases, better preserve antigens but will produce a higher background and impact on structural integrity. Therefore, it is recommended to test different protocols for a “new” protein or epitope – the results will pay back your investment.
Key wordsActin focal adhesion microtubule intermediate filament extracellular matrix fixation by cross-linking fixation by precipitation myofibroblast wound healing fibrosis
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