Immunocytochemical localization of the glucocorticoid receptor in rat brain, pituitary, liver, and thymus with two new polyclonal antipeptide antibodies.

0013-7227/91/1296-3064$03.00 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 6 Printed in U.S.A.

Immunocytochemical Localization of the Glucocorticoid Receptor in Rat Brain, Pituitary, Liver, and Thymus with Two New Polyclonal Antipeptide Antibodies* WILLIAM C. McGIMSEY, JOHN A. CIDLOWSKI, WALTER E. STUMPF, AND MADHABANANDA SAR Departments of Cell Biology and Anatomy (W.C.M., W.E.S., M.S.) and Physiology (J.A.C.), University of North Carolina, Chapel Hill, North Carolina 27599

ABSTRACT. The intracellular localization of the glucocorticoid receptor (GR) was studied in male rat brain, pituitary, liver, and thymus. Two new polyclonal anti-GR antibodies, GR 57 and GR 59, raised against two synthetic peptides (346-357 and 245-259) that correspond to unique regions of the amino-terminus of human GR were used. Vibratome sections (30-50 fim) of perfused brain and frozen sections (6-8 fim) of pituitary, liver, and thymus fixed in paraformaldehyde were incubated in preimmune serum, immunoserum, epitope-purified immunoserum, or peptide-absorbed immunoserum of either GR 57 or GR 59 and immunostained by the avidin-biotin peroxidase method. GR immunoreactivity (GR-ir) was primarily nuclear in brain, pituitary, liver, and thymus sections from intact rats. Adrenalectomy caused nuclear GR-ir to decrease and cytoplasmic GR-ir to increase. When adrenalectomized rats were treated with corticosterone (100 ng and 1 mg) or dexamethasone (1 Mg. 100 Mg> and 1 mg), GR-ir was again predominantly nuclear. One micro-

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HE GLUCOCORTICOID receptor (GR) is a member of a superfamily of ligand-dependent nuclear transcription factors (1). Several monoclonal antibodies raised against GR isolated from rat liver have been produced (2-4) and used for cellular and subcellular localization of GR (5-7). Recent cloning and sequencing of steroid hormone receptors has enabled investigators to raise antibodies against synthetic peptides specific to steroid hormone receptor proteins. Antipeptide antibodies raised to sequences corresponding to the DNA-binding domain have been shown to recognize only the activated form of the progesterone receptor (8) and GR (9). Other sequence-specific antibodies have been raised against peptides corresponding to the N -terminus region of some steroid hormone receptors. These antibodies

Received July 8,1991. Address all correspondence and requests for reprints to: Madhabananda Sar, Ph.D., Department of Cell Biology and Anatomy, University of North Carolina, 526 Taylor Hall, CB 7090, Chapel Hill, North Carolina 27599. * This work was supported by NIH Grant NS-17479 and P30-HD18968 (Histochemistry Core).

gram of corticosterone failed to cause nuclear GR-ir when administered to adrenalectomized rats. Immunoreactive neurons and glial cells were extensively distributed, with varied intensity, throughout the rat forebrain. The areas include cortex, septum, hippocampus, amygdala, thalamus, and hypothalamus. Cells with the strongest GR-ir were located in the caudate putamen, paraventricular, arcuate, and central amygdala nuclei, areas CA1-CA2 of the hippocampus, and laminae 4 and 5 of the cortex. In the pituitary, cells of the anterior and posterior lobes were GR immunoreactive, while those in the intermediate lobe were not. Hepatocytes of the liver and thymocytes and reticuloepithelial cells of the thymus were GR immunoreactive. The results show that GR can be localized immunocytochemically in numerous rat tissues using antipeptide polyclonal antibodies and correlated with the results of biochemical and ligand receptor studies. {Endocrinology 129: 3064-3072,1991)

have been used to study, immunocytochemically, the cellular distribution of androgen receptor in rat reproductive organs, pituitary, brain, and human tissue (10, 11), that of mineralocorticoid receptor in rat and human kidney (12,13), and that of GR in rat pituitary (14). Most recently, two synthetic peptides that correspond to unique regions of the amino-terminus of human GR, 346-367 and 245-259, have been used to produce epitopespecific antibodies GR 57 and GR 59, respectively. Sucrose density gradient analysis of the receptor-antibody complexes and immunoassay showed that both anti-GR antibodies recognize nonactivated and activated forms oftheGR(15). There are a number of issues that can be addressed immunocytochemically with these new anti-GR antibodies. The immunocytochemical intracellular localization of GR in rat brain and liver, in the presence or absence of hormone, with different anti-GR antibodies has not yielded consistent results (5, 7,16,17). Autoradiographic studies of the localization of [3H]corticosterone and [3H] dexamethasone in the rat brain showed different distri-

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NUCLEAR AND CYTOPLASMIC GR LOCALIZATION

butions (18-20), probably reflecting a dose-dependent occupation of the GR (21). Although there is extensive biochemical evidence for the existence of GR in the thymus, it has not yet been immunocytochemically demonstrated. The present studies, using GR 57 and GR 59, were designed to assess the distribution of GR immunoreactivity (GR-ir) in rat brain, pituitary, liver, and thymus of the intact rat, observe the subcellular location of GR in target tissues in the presence or absence of glucocorticoid hormones, and show the dose dependence of corticosterone and dexamethasone on the nuclear localization of GR-ir.

Materials and Methods Animals Adult male Sprague-Dawley rats (150-200 g) were used in all experiments. The animals were kept under standard environmental conditions (lights on from 0500-1900; temperature, 22 C; relative humidity, 70%) and received lab chow and water ad libitum. Animal treatment Some rats were kept intact; others were adrenalectomized bilaterally and maintained on saline (0.9%) drinking water. Four days after adrenalectomy, the animals received a single injection of corticosterone (1 ng, 100 ng, or 1 mg/100 g BW, ip) or dexamethasone (1 ng, 100 ng, or 1 mg/100 g BW, ip) in 0.2 ml 70% ethanol. The control adrenalectomized rats received only vehicle. One hour after corticosterone or 6 h after dexamethasone administration, the rats were perfused with fixative. Chloral hydrate (4% in saline; 1 ml/100 g BW, ip) was administered as an anesthetic agent before adrenalectomy or perfusion. At least six animals were used in each experimental condition, except for the thymus, where three rats were used. Tissue preparation Anesthetized animals were perfused through the heart (ascending aorta) with 200 ml PBS (pH 7.4), followed by 400 ml 4% paraformaldehyde plus 0.2% glutaraldehyde (pH 7.4). The brains were removed, postfixed in 4% paraformaldehyde for 4 h, and stored in PBS at 4 C overnight until they were sliced into 30- to 50-/xm thick sections on a vibratome for immunostaining. All perfusions were performed in the afternoon. Whole pituitaries and pieces of liver and thymus were quickly removed from rats that had been decapitated. The tissue was frozen onto brass holders in 2-methylbutane cooled in liquid nitrogen (-180 C); the frozen tissue was stored in liquid nitrogen. Six- to 8-/xm sections were cut in a cryostat. The sections were picked up on gelatin-coated glass slides, air dried for 5 min, fixed in 4% paraformaldehyde (pH 7.4) plus 10% sucrose for 5 min, then immunostained. The thymus was removed from three additional 150-g rats. Each thymus was finely minced with a razor in a small volume of PBS. A smear of the suspension was made on a glass slide, air dried for 5 min, fixed in 4% paraformaldehyde, then proc-

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essed for immunocytochemistry by the avidin-biotin peroxidase method and the immunofluorescence technique. Immunocytochemistry The immunocytochemical method used has been described previously (22). Immunosera and epitope-purified immunoesera of GR 57 (1.1 mg/ml) or GR 59 (2.8 mg/ml) were used at a dilution of 1:5000 on the vibratome sections, and purified immunoserum GR 57 (1.1 mg/ml) at a dilution of 1:1000 was used on the fixed frozen sections. The vibratome sections were incubated in primary antibody diluted with PBS containing 2% normal goat serum and 0.2% Triton X-100 at 4 C for 24 h. The cryostat sections were put in 0.2% Triton X-100 in PBS for 5 min, then incubated in 2% normal goat serum and primary antibody for 12-24 h at 4 C in a humid chamber. Vectastain, ABC Kit (Vector Laboratories, Inc., Burlingame, CA) reagents were used at the following dilutions: secondary antibody (biotinylated goat antirabbit), 1:200; and avidin-biotin peroxidase complex, 1:400 (for 1 h each). Freshly prepared diaminobenzidine (50 mg/100 ml PBS) containing 8 i*\ 30% H2O2 was used as the chromagen. The thymus smears were put in 0.2% Triton X-100 in PBS for 5 min, then incubated in 2% normal goat serum and primary antiserum for 12-24 h at 4 C in a humid chamber and stained by the avidin-biotin peroxidase method described above. For immunofluorescence, the smears were treated with 0.2% Triton X-100, followed by incubation with 5% BSA in PBS for 25 min to block nonspecific staining. The smears were incubated with purified GR 57 at a dilution of 1:400 at 4 C. After overnight incubation, the smears were washed in PBS and then treated with fluorescein isothiocynateconjugated goat antirabbit immunoglobulin G (1:400; Organon Technika, Cochranville, PA) for 1 h at room temperature. The slides were washed in PBS, mounted with polyvinyl alcoholglycerol, coverslipped, and photographed using an Olympus fluorescence microscope (New Hyde Park, NY). Some GR-stained brain sections were counterstained with aqueous toluidine blue to facilitate identification of nuclear groups. The stained sections were then mounted on glass slides, air dried, and coverslipped with Permount. Photographs were taken using an Olympus BH-2 microscope. In intact rat brain cells the GR-ir was considered strong if the cell nucleus was dark brown and distinct from any cytoplasmic or background stain. Moderate immunostaining consisted of cells in which the nucleus was distinct from cytoplasmic or background stain, but was light brown in color. Weak immunostaining consisted of a very light brown staining in both the cytoplasm and nucleus. Cells with no GR-ir, detected only when counterstained with toluidine blue, had light blue cytoplasm and clear nuclei. Preparation of antisera GR 57 and GR 59 and their purification by affinity chromatography have been previously described (15). The specificity of immunostaining in vibratome and frozen sections was established by incubating the sections with preimmune serum, absorbed immunoserum, or absorbed epitope-purified immunoserum. Absorption involved mixing the immunoserum with the appropriate peptide immunogen. Ten to 50 micrograms of peptide were mixed with 1 ml antiserum, 1:5000 for vibratome sections and 1:1000 for frozen sections, before incubation of tissue in immunoserum.


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Results Immunocytochemical experiments demonstrated specific immunostaining for anti-GR antibodies GR 57 and GR 59 in intact rat brain (Fig. 1, A and B) and liver (Fig. 2, A and B). Vibratome brain sections (1:5,000) and cryostat liver sections (1:1,000) incubated with immunoserum or epitope-purified serum exhibited primarily nuclear GR-ir (Figs. 1, A and B, and 2, A and B). Dilutions greater than 1:15,000 on brain sections or 1:3,000 on liver sections lacked immunostaining (not shown). Incubation of brain and liver sections in absorbed immunoserum or absorbed epitope-purified immunoserum resulted in the absence of immunostaining (Figs. 1, D and E, and 2D). Sections of brain and liver incubated in preimmune serum had no specific immunostaining (Figs. 1C and 2C). Brain The distribution of GR-ir in the rat brain was similar for GR 57 and GR 59. The subcellular location of GR-ir in strongly stained cells for GR 57 and GR 59, serum or

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epitope purified, was principally nuclear (Fig. 3A). Four days after adrenalectomy the GR-ir had increased in the cytoplasm and had decreased substantially in the nuclei (Fig. 3B). Nuclear GR-ir was reestablished in adrenalectomized rats given either corticosterone (100 fig 1 mg/ 100 g BW; Figs. 3C and 4C) or dexamethasone (1 fig, 100 ixg, or 1 mg/100 g BW; Figs. 3D and 4, B and D). Glial cells nuclei showed moderate to dark staining (not shown). GR translocation was dose dependent. The larger doses of corticosterone or dexamethasone administered to adrenalectomized rats resulted in a greater decrease in cytoplasmic GR-ir and an increase in nuclear GR-ir in pyramidal cells of the area CA1 of the hippocampus. A dose of 1 fig corticosterone showed cytoplasmic GR-ir with little or no nuclear GR-ir (Fig. 4A). A dose of 1 fig dexamethasone caused moderate nuclear GR-ir; a light cytoplasmic stain was also evident (Fig. 4B). The low dose of dexamethasone affected those cells that contained the darkest GR stain in the intact rat, i.e. CA1CA2 regions of the hippocampus. A dose of 100 ^tg or 1 mg corticosterone (Figs. 4C and 3C) or dexamethasone

FIG. 1. GR localization in rat hippocampus. Vibratome sections were immunostained with antiserum GR 57 (A), GR 59 (B), preimmune serum (C), absorbed antiserum GR 57 (D), and absorbed antiserum GR 59 (E). Note the absence of immunostaining in pyramidal cells of area CA1 with preimmune serum and absorbed serum, and the specific nuclear staining with immunoserum. Sections were 50 urn thick. Magnification, X485.

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FIG. 2. GR localization in rat liver. Sections were incubated with purified GR 57 (A and B), preimmune serum (C), and absorbed antibody GR 57 (D). Note the dark nuclear stain (A and B) and the absence of nuclear immunostaining in hepatocytes (C and D). Sections were 6 ^m thick. Magnifications: A, X78; B, C, and D, X312.

FlG. 3. GR localization in pyramidal cells of area CAl of the hippocampus. Note nuclear immunostaining in intact rat (A), increased cytoplasmic staining and decreased nuclear staining in sections from adrenalectomized rats (B) and from adrenalectomized rat treated with 1 mg corticosterone (C) or dexamethasone (D). Sections were 50 lira thick. Magnification, X312.

(Figs. 3D and 4D) induced nuclear GR-ir in more cells than the l-/ug dose (Fig. 4, A and B). At the highest doses, no cytoplasmic GR-ir, but strong nuclear GR-ir, was detectable. GR-ir was widely distributed throughout the intact rat forebrain (Fig. 5) and exhibited a wide variety of immunostain intensities in brain nuclei, ranging from dark to light. Four levels of rat brain in the frontal plane (cor-

responding to Figs. 15, 20, 25, and 27 of Ref. 23) were examined as a sample of the extensive distribution and variation of intensities of the GR immunostaining in the intact rat forebrain. At the most rostral level examined, most cells of the caudate putamen, nucleus accumbens, and claustrum contained dark-staining nuclei. In cerebral cortex many neurons, especially in laminae 4 and 5, showed strong


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FIG. 4. GR localization in pyramidal cells of area CAl of the hippocampus from adrenalectomized rats treated with 1 ng corticosterone (A) or dexamethasone (B), 100 fig corticosterone (C), or dexamethasone (D). Note the cytoplasmic stain with little or no nuclear stain in cells with the low dose of corticosterone compared to the primarily nuclear stain seen with the higher doses. Sections were 50 nm thick. Magnification, X160.

FIG. 5. Immunocytochemical GR distribution in intact male rat brain. Vibratome sections were stained with purified GR 57 antibody. A: CTX, Cortex; H, hippocampus. B: AM, Amygdala and piriform cortex. C: Anterior hypothalamic area including paraventricular nucleus (PN). D: Central hypothalamus, showing the arcuate (AR), ventromedial VM, and dorsomedial nuclei (DM). Note the strong immunostaining in CAl area of the hippocampus (A), central amygdala nucleus (CN; B), and parvocellular portion of paraventricular nucleus (PN). Sections were 50 /xm thick. Magnification, X28.

GR-ir in nuclei. The rest of the cortical cells contained moderate levels of GR-ir (Fig. 5A). The piriform cortex contained moderately stained cells (Fig. 5B). The lateral septal nucleus consisted of a mixture of strong, moderate, and weakly GR-positive neurons, while the medial septal nucleus consisted only of weakly immunoreactive cells. At the next level, the bed nucleus of the striae termi-

nalis contained dark-staining cells, while many cells of the preoptic nuclei and the hypothalamus contained weakly GR-immunoreactive cells. At the two caudal levels, most neurons in the hypothalamus contained a low intensity of GR-ir; the exceptions were some moderately stained cells of the anterior hypothalamic area and dark, intensely stained cells in



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the parvocellular paraventricular (Fig. 5C), ventromedial, and arcuate nuclei (Fig. 5D). Hippocampal pyramidal cells of areas CA1 and CA2 consistently showed strong immunoreactivity (Fig. 5A), while with different animals, cells of area CA3 ranged from moderate to weak GR-ir. The granule cells of the dentate gyrus contained moderately stained nuclei, the reticular thalamic nucleus and the zona incerta contained weakly stained cells, and the ventral and lateral thalamic nuclei darkly stained cells. Midline thalamic cells had a moderate to weak amount of nuclear GR-ir. In the amygdala (Fig. 5B), strong GR-immunoreactive neurons were located in the central and medial nuclei, while cells of basolateral, basomedial, and anterior cortical nuclei contained medium staining intensity. Cells of the supraoptic nucleus and magnocellular paraventricular nucleus contained no GR-ir and were visible only when counterstained with toluidine blue. Pituitary, liver, and thymus Anterior and posterior lobe pituitary cells of intact rats exhibited dark nuclear immunostaining (Fig. 6, A, B, and C). Approximately 60-80% of anterior lobe pituitary cell had nuclear GR-ir. Nuclear GR-ir was substantially reduced and cytoplasmic stain increased in pituitaries of adrenalectomized rats (not shown). Intermediate lobe cells contained no specific GR-ir (Fig. 6A). Incubation of pituitary sections with preimmune serum resulted in no specific staining (Fig. 6D). In sections from intact rat liver all hepatocytes showed primarily nuclear GR-ir (Fig. 7A). The immunostain of livers from adrenalectomized rats showed that nuclear GR-ir was absent, while cytoplasmic staining was evident in hepatocytes (Fig. 7B). Some thymic reticulo-epithelial cells and thymocytes displayed nuclear GR in immunostained thymus sections from intact rats (Fig. 8A) and immunofluorescent stained smears of minced thymus (Fig. 9) Thymus sections incubated with preimmune serum showed no specific staining (Fig. 8B).

Discussion The two new anti-GR antibodies GR 57 and GR 59 proved useful in addressing several questions regarding GR in rat tissue. In the darkest staining neurons of the intact male rat brain, GR-ir was principally nuclear with some cytoplasmic staining. After adrenalectomy nuclear GR-ir was decreased, while cytoplasmic GR-ir was increased. When hormone was replaced, strong nuclear GR-ir was reestablished, GR translocated from the cytoplasm to the nucleus. This pattern of subcellular localization of GR-ir was also seen with GR 57 in cultured Hela S3 cells and

FIG. 6. GR localization in pituitary of intact male rat. Sections were stained with purified GR 57 antibody. Note the strong immunostaining in nuclei of anterior lobe (AL) cells (A and B) and posterior lobe (PL) cells (A and C) and the lack of specific immunostaining in intermediate lobe (IL) cells (A). With preimmune serum no immunostaining was seen in anterior (AL), intermediate (IL), or posterior (PL) lobe cells (D). Sections were 8 /zm thick. Magnifications: A and D, X150; B, X450; and C, X300.

Chinese hamster ovary cells (15). Several investigators using different monoclonal and polyclonal antibodies to GR reported similar results on GR-ir in the rat brain (5), liver (24), and monolayer of cultured cells (25-29). However, some immunocytochemical studies (7, 16, 17) have shown the GR to be nuclear in the presence or absence of hormone. Future studies directly comparing GR-ir seen among different anti-GR antibodies may resolve the discrepancies. Since glucocorticoid effects on the brain are receptormediated events, these are the possible sites for the


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FIG. 7. GR localization in liver of intact rat (A) and adrenalectomized rat (B). Note immunostaining in nuclei of hepatocytes in intact rats and the absence of nuclear staining but the appearance of cytoplasmic staining in the section from the adrenalectomized rat. Sections were 6 jim thick. Magnification, X410.

central action of glucocorticoids. Glucocorticoids have been shown to have numerous effects on brain function, including regulation of pituitary hormone secretion (30), learning and memory (31), electrophysiological activity (32), and sensory functions (33). The distribution of GR-

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ir demonstrated in the rat brain is in general agreement with in situ hybridization histochemistry of GR mRNA levels (34, 35), GR immunocytochemistry (5, 6), and [3H] dexamethasone autoradiography (18) (Stumpf, W. E., and M. Sar, unpublished observations). The range of intensity of GR-ir detected with GR 57 correlates with [3H]dexamethasone autoradiographic studies, which have shown that nuclear accumulation of radioactivity varies considerably in different regions of the brain (18) (Stumpf, W. E., and M. Sar, unpublished observations). In the hippocampus strong nuclear GR-ir and strong nuclear labeling with [3H]dexamethasone were found in the CA1 pyramidal cells. Granule cells of the dentate gyrus and the CA3 pyramidal cells displayed moderate to weak GR-ir and [3H] dexamethasone radiolabeling. Although immunocytochemistry is only semiquantitative, the relative intensities of GR-ir probably reflect the amount of GR within a cell. A cell's responsiveness to glucocorticoids is dependent upon the amount of GR (36). Thus, cells with the strongest GR-ir, such as CA1 pyramidal hippocampal cells, show GR nuclear translocation at the lowest dose of dexamethasone. CA3 pyramidal hippocampal cells, which contained little to moderate intensity nuclear GR-ir, are vulnerable to high levels of glucocorticoids (37), a phenomenon thought to be mediated by GR (38). Dentate gyrus cells, with only moderate nuclear GR-ir intensity, are known to require glucocorticoids for survival (39). Therefore, even cells with minimal GR-ir are strongly influenced by glucocorticoids. The autoradiographic localization of [3H]corticosterone and [3H]dexamethasone in the rat brain (18-20) differed, probably because a dose of 1 jtg/100 g BW [3H] corticosterone was only enough steroid to saturate the mineralocorticoid receptor (21), which has high affinity for both corticosterone and mineralocorticoids. The distribution of [3H]corticosterone and [3H]aldosterone in the rat brain are similar (40). In this study it was observed that 1 ng dexamethasone, but not 1 ng corticos-

FiG. 8. GR localization in thymus. Sections were immunostained with GR 57 antibody (A) and preimmune serum (B). Reticuloepithelial cells as well as thymocytes displayed nuclear immunostaining (A). With preimmune serum no specific staining could be detected (B). Sections were 8 Mm thick. Magnification, X475.



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chemistry and immunofluorescence, with smears of cells from minced thymus. In further studies specific thymic cell types can be isolated with careful dissociation techniques (53, 54). Thymic reticuloepithelial cells contain multiple steroid hormone receptors. Colocalization studies using autoradiography and immunocytochemistry (41) or immunocytochemistry with antibodies to different steroid hormone receptors can establish the extent of overlap of steroid hormone receptors in reticuloepithelial cells. FlG. 9. Immunofluorescent localization of GR in cell nuclei from a smear of minced thymus. Magnification, X333.

References

terone, caused nuclear localization of the GR. Therefore, 1 ng [3H]corticosterone would not be a sufficient dose to autoradiographically detect nuclear radioligand GR. With combined autoradiography and immunocytochemistry (41), localization of ligand and receptor in the same cells can be useful in clarifying the sites of action of steroid hormones. GR-ir was localized in cells of the anterior and posterior lobes of the pituitary. This is consistent with previous [3H]dexamethasone autoradiography studies (4244) and immunohistochemical localization of GR in the pituitary (14). Immunocytochemical colocalization studies with antibodies to anterior pituitary hormone and GR can be performed to determine which pituitary cells contain GR-ir. Liver sections showed nuclear GR-ir in hepatocytes in intact rats, while cytoplasmic staining was detected in untreated adrenalectomized rats. Using two different monoclonal antibodies raised against partially purified GR proteins from rat liver, Gasc et al. (16, 17) reported nuclear localization of GR in paraffin and frozen sections of rat liver 1-2 weeks after adrenalectomy. This result contradicts our finding in rats adrenalectomized for 4 days. Methodological differences and adrenal tissue regeneration may account for the discrepancies. Effects of glucocorticoids, adrogens, and estrogen on the thymus are well known. Estrogen receptor, androgen receptor, and progesterone receptor have been biochemically demonstrated in thymic reticuloepithelial cells, but not in lymphocytes (45-47). GR within rat thymocytes has been reported (48). Autoradiographic studies with [3H]estradiol (49, 50) and [3H]l,25-dihydroxyvitamin D3 (51, 52) have shown nuclear labeling in reticuloepithelial cells, but not in thymocytes. Immunocytochemical staining of mineralocorticoid receptor has been reported in a few nonlymphatic cells (12,13). Our observations are the first immunocytochemical demonstration of GR in thymocytes and reticuloepithelial cells, substantiating the biochemical detection of GR in thymus. The difficulty of staining for GR in frozen thymus sections indicates the need for additional immunostaining, both immunocyto-

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Monoclonal antipeptide antibodies to the glucocorticoid receptor.

Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies.

Polyclonal antibodies against the rat NK1 receptor: characterization and localization in the spinal cord.

Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors.

Immunocytochemical localization of apolipoprotein A-I using polyclonal antibodies raised against the formaldehyde-modified antigen.

Immunocytochemical localization of argininosuccinate synthetase in the rat brain.

Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform.

Antipeptide antibodies against the 5-HT1A receptor.

Immunocytochemical localization of growth hormone receptor in rat maxillary teeth.

Immunocytochemical localization of endopeptidase 24.15 in rat brain.

Immunocytochemical localization of phosphatase inhibitor-1 in rat brain.

Glucocorticoid binding to receptor-like proteins in rat brain and pituitary: ontogenetic and experimentally induced changes.

Glucocorticoid-receptor interactions in rat liver.

Immunocytochemical localization of aromatase in the brain.

Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor.

Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate.

Immunocytochemical localization of N-acetyl-aspartate with monoclonal antibodies.

Immunocytochemical localization of alpha-fetoprotein in the developing and carbon tetrachloride-treated rat liver.

Vitamin A deficiency and glucocorticoid receptor activity in rat liver.

Light and electron immunocytochemical localization of AMPA-selective glutamate receptors in the rat brain.

Opiate receptor: autoradiographic localization in rat brain.

Angiotensin receptors from rat liver, brain and pituitary gland. Expression of two subtypes in Xenopus oocytes.

Stress-induced changes of glucocorticoid receptor in rat liver.

Immunocytochemical localization of calretinin in the forebrain of the rat.