Detection of insulin by immunocytochemistry is one of the most powerful and sensitive techniques available to monitor levels of expression in islets (1), β-cells in culture (2), tissue samples (3), and cells transgenically expressing the insulin gene (4). Like all immunocytochemistry techniques, much depends on the availability of a high-quality primary antibody. Species-specific insulin antibodies are now available from a number of commercial sources (including Santa Cruz), and these allow sensitive and specific detection of even very low levels of expression. Described below is the protocol for detection of insulin in β-cell lines in culture, using a FITC-coupled secondary antibody. A broad range of secondary antibodies are also available, including fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and biotin-labeling, among others. For cells in culture, two-well, four-well, or eight-well chamber slides are recommended (available from several commercial sources, including Nunc), as these allow rapid and simple processing of large numbers of replicate samples, with small volumes of antibody. However, the following protocol can also be applied to cells grown on coated cover slips. The methods described here are for analysis by fluorescence microscopy. For analysis of insulin expression in intact islets, confocal microscopy would be required (1).
|Title of host publication||Diabetes Mellitus|
|Subtitle of host publication||Methods in Molecular Biology|
|Number of pages||3|
|Publication status||Published - 2003|
|Name||Methods Mol Med|