In situ hybridization is used to localize mRNA in brain regions or cell types. The radioactive version is a sensitive assay for which cell type expresses which gene; the use of digoxygenin-labelled probes is less sensitive but permits double labelling for multiple gene expression. There is good agreement between different labs on the results with GABAA receptor gene expression, probably because hybridization of nucleic acids is reasonably standardized between labs. The disadvantage of in situ hybridization is that no information is obtained on protein localization on the cell, or even if the mRNA is translated. Thus immunocytochemistry with subunit-specific antibodies gives the ultimate biological information. However, antibodies can be problematic; Saper and Sawchenko (2003) point out that antibodies are biological agents, not standard chemical reagents: antibodies may bind to a wide variety of antigens other than the one that they were raised to recognize and there is no way to be sure that the pattern they stain really represents that antigen. If investigating localization in the rodent nervous system, a knockout mouse for the particular antigen is the best control of antibody specificity (Saper and Sawchenko, 2003; Aller et al., 2005). This has been done for some (e.g., al, a3, a6 and 8) (Jones et al., 1997; Tretter et al., 2001; Yee et al., 2005; Kralic et al., 2006), but not all GABAa receptor antibodies used in published papers.
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