Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique

Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60 degrees C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cells transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation.