Specificity of glucocorticoid receptor primary antibodies for analysis of receptor localization patterns in cultured cells and rat hippocampus

Abstract

After glucocorticoid stimulation, glucocorticoid receptors (GRs) are translocated to the nucleus to modulate transcription of glucocorticoid target genes. The subcellular distribution and trafficking of GR in cultured cells has been studied quite intensively using several techniques. However, the intracellular localization of nuclear receptors in ligand-free and stimulated conditions in vivo is still controversial, in part because of inconsistent results with different antibodies. Knowledge of trafficking of GR in vivo could greatly contribute to understanding nuclear receptor signaling. Therefore, in this study we systematically compared a panel of different primary GR antibodies using immunohistochemistry and confocal imaging. Nuclear translocation patterns at different time points after glucocorticoid stimulation were compared in cultured AtT20 cells and rat hippocampal CA1 and dentate gyrus cells. The BuGR2 antibody consistently detected GR nuclear translocation patterns between in vivo and in vitro settings, but the other GR primary antibodies provided contradictory results. While GR H300 and P20 strongly detected nuclear GR immunoreactivity after glucocorticoid stimulation in both CA1 and dentate gyrus cells, the same antibodies provided poor results in cultured cells. The opposite was found for the primary GR M20 antibody. These data indicate that with a particular glucocorticoid receptor antibody the findings in cell culture studies cannot always be extrapolated to in vivo situations. Moreover, different antibodies disclose different features of the glucocorticoid receptor translocation process.