ARTICLE |
Correspondence to: Mariann Fodor, Dept. of Pediatrics, Vrije Universiteit Medical Center, 1007 MB Amsterdam, The Netherlands. E-mail: fodor@vumc.nl
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Summary |
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Difficulties in demonstrating sex steroid receptors in the human brain by immunohistochemistry (IHC) may depend on postmortem delay and a long fixation time. The effect of different postmortem times was therefore studied in rat brain kept in the skull at room temperature for 0, 6, or 24 hr after death. After a long fixation for 20 days, hypothalami were embedded in paraffin and sections were immunohistochemically stained for androgen receptor (AR), estrogen receptor- (ER), or progesterone receptor (PR). Retrieving the antigenic sites by microwave pretreatment was essential to obtain successful IHC in all groups studied. In general, immunoreactivity was restricted to the cell nuclei. However, the intensity of the staining appeared to be strongly dependent on the different receptor antigens and postmortem time. Both AR and ER but not PR immunoreactivity were decreased after immersion-fixation compared to the perfused sections at time point zero. In brains fixed by immersion, all three receptors decreased gradually with increasing postmortem time, and ER became hardly detectable after 24 hr postmortem. The results of these experiments show that, with the protocol used, postmortem variables and lengthy fixation do not, in principle, prevent sex steroid receptor IHC in human material. The outcome of the immunostaining, however, might be strongly dependent on the epitopes and/or antibody used. (J Histochem Cytochem 50:641649, 2002)
Key Words:
estrogen receptor-, androgen receptor, progesterone receptor, immunohistochemistry, postmortem delay, rat
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Introduction |
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THE GONADAL STEROID HORMONES testosterone, estrogens, and progesterone play a critical role in the structural and functional development of the central nervous system, resulting, e.g., in sex differences in sexual and reproductive behavior and neuroendocrine processes. Sex steroids mediate their function by binding to the appropriate intracellular receptors that belong to the superfamily of ligand-responsive transcription factors (
The effect of postmortem delay on steroid receptors is strongly dependent on the technique used to visualize them. In spite of the labile receptor binding properties of corticosteroids during a postmortem period of 34.5 hr (
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Material and Methods |
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Animals and Tissue Preparation
Male Wistar rats were purchased from Harlan CPB (Zeist, The Netherlands). All aspects of animal care and treatment were carried out according to the local guidelines of the experimental animal care committee (No. 95.19/00). Rats weighing 200 ± 20 g (n=20) were sacrificed by decapitation and the brains were either removed immediately (n=5) or left in the skull for 6 (n=5) or 24 h (n=5) at room temperature (RT). For comparison, five intact animals were deeply anesthetized with sodium pentobarbital (0.1 ml/100 g body weight) and perfused through the ascending aorta with 50 ml of physiological saline, followed by 300 ml of fixative containing 4% paraformaldehyde in 0.1 M PBS, pH 7.4. All groups were then treated as described below. After a postmortem delay of 0, 6, or 24 hr the brains were removed from the skull and postfixed for 20 days in 4% paraformaldehyde solution in 0.1 M PBS at 4C. The forebrains were processed for routine paraffin (Histowax) embedding. Coronal sections (8 µm) were cut, mounted on 3-amynopropyltriethoxisilane-coated slides (Menzel; Darmstadt, Germany) to prevent detachment due to the high temperature during antigen retrieval treatment. Antigen retrieval was performed by microwave oven heating according to
Immunohistochemistry
After rehydration, the sections were first rinsed extensively with PBS and incubated overnight in one of the primary antibodies diluted in Tris-buffered saline (TBS) containing 0.05 M Tris, 0.5 M NaCl, 0.5% Triton X-100, and 0.1% sodium azide, pH 7.6, at 4C. The primary antibodies included PG 21 (1:500) (gift of Geoffrey Greene, University of Chicago), which is a rabbit polyclonal androgen receptor (AR) antibody directed at the first 21 amino acids at the N-terminus of the rat/human AR () antibody raised against amino acids 118140 in the A/B domain of the human ER (
Quantitative Analysis
To determine the postmortem effects in the staining intensity of AR/ER/PR-containing neurons, immunoreactivity was evaluated by optical density measurements. Brain regions rich in a given receptor subtype were studied, including the lateral septum (AR), arcuate nucleus (ER, PR), and medial preoptic nucleus in which all three receptors (AR, ER, PR) were present. Two sections per cell group per animal were analyzed for a total of 100 sections using an IBAS image analysis system (Kontron Instruments; Munich, Germany). Sections were viewed under brightfield illumination with a Zeiss Axioplan microscope with a SONY CCD XC-77CE black-and-white camera. Gain and black level of the camera were fixed. A place outside the tissue was chosen for 100% transmission (gray level 255).
The same illumination and analog settings were used during acquisition and analysis of all images. The gray values per pixel were transformed into optical density (OD) per pixel and average OD per cell was measured. For each cell group from each animal, OD readings from two sections (20 cells) were summed. To analyze background staining, five OD readings per section were taken from a cell-sized area of neuropil in the vicinity of labeled neurons.
The differences between the intensity of the immunohistochemical staining for each time point was analyzed by ANOVA tests. Differences were considered statistically significant at p<0.05.
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Results |
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Paraffin-embedded sections of rat forebrain showed specific nuclear labeling for all three sex steroid receptors studied, in spite of the long fixation period of 20 days. However, for visualization of the immunoreactivity microwave irradiation was essential. An example is given in Fig 1, in which the lateral septum (AR-containing region) and the neighboring caudate putamen complex (AR-negative region) are shown. In general, sections without microwave pretreatment showed nonspecific reaction in the ependymal layer of the ventricular system, high background, and absence of specific nuclear staining all over the section (Fig 1A), in contrast to the microwave-pretreated sections, in which specific nuclear staining became visible only in the expected area and the ependymal staining disappeared (Fig 1B).
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Androgen Receptor Immunoreactivity
Cells containing AR immunoreactivity were widely distributed throughout the intact rat forebrain. The highest number of cells was found in the lateral septum (Fig 1), the bed nucleus of the stria terminalis, amygdala, medial preoptic area, and the hypothalamic ventromedial, arcuate, and premamillary nuclei. Immunopositivity was restricted to the cell nuclei as was shown previously in immunohistochemical staining in the gonad-intact male (
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Estrogen Receptor Immunoreactivity
ER immunoreactivity was distributed as previously described (
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Progesterone Receptor Immunoreactivity
At time point zero, PR immunoreactivity was observed in cell nuclei of neurons in the preoptic area and mediobasal hypothalamus (Fig 3) as previously described (
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Discussion |
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This is the first report evaluating postmortem effects on sex steroid receptor immunohistochemistry in paraffin-embedded rat brain. In recent years, several studies have indicated that immunohistochemical demonstration of sex steroid receptors in the brain of different species, including humans, is feasible. The descriptive studies in mammalian brain, with one exception (
For systematic, retrospective, and quantitative work on the human brain, however, the most convenient way to collect brain material is to embed the tissue block in paraffin after appropriate fixation. Experimental animals are mostly transcardially perfused with the fixative, whereas human autopsy material is usually immersion-fixed. Working with human tissue usually involves additional unfavorable factors such as the variable postmortem delay, no perfusion, and therefore inadequate fixation because fixation of the outer parts of the tissue blocks forms a barrier. As a consequence, a longer fixation time is necessary for the larger human brain structures (
The aim of our study was to see whether postmortem delay, independently of other factors, might affect sex steroid receptor immunohistochemistry. For that purpose we used intact adult male rats because we wanted to avoid cyclic changes in the hormonal milieu.
To detect the steroid receptor proteins in the paraffin-embedded rat brain it was necessary to pretreat the samples to open the tissue matrix and to minimize the crosslinkage caused by the aldehyde fixative (
Ischemia after decapitation may lead to serious deleterious effects on receptor staining at the protein level. Loss of immunoreactivity was observed in the estrogen and androgen receptor immunostaining compared to perfusion data. These results indicate that ischemic effects are dependent on receptor types and/or antigen epitopes. It is important to note, however, that in all groups immunohistochemically stained for ER, AR, or PR, the distribution pattern of immunopositive cells did not appear to differ between these two conditions at the zero time point. Although sex steroid receptor-containing nuclei were still visible 6 hr after death, the immunostaining was gradually and markedly decreased. ER became hardly detectable after a 24-hr postmortem delay. Weakly stained PR-immunoreactive cell populations were still visible 1 day after death, but the staining was less homogeneous due to strongly labeled single neurons. In comparison to ER and PR, however, after 1 postmortem day a less dramatic decrease in the AR immunoreactivity was observed. Taken together, ER and PR immunostaining was most prominently influenced by the postmortem time.
Speculation that the length of fixation might result in cytoplasmic staining, as was reported earlier on free-floating material (
Taken together, postmortem delay and long fixation time are believed to prevent successful immunohistochemical staining of steroid receptors. The present study shows that the detrimental effect of prolonged fixation can be effectively reversed by microwave treatment. Although immunoreactivity of sex steroid receptors does not immediately deteriorate due to postmortem delay, the quality of the staining is very much dependent on antigens and antibodies.
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Acknowledgments |
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Supported by NWO (#903-47004) to MF.
We thank Drs Gail S. Prins and Geoffrey L. Greene (University of Illinois, Chicago, IL) for the PG-21 antibody, Dr C. W. Pool and Mr J. J. van Heerikhuize for the IBAS program, Dr M. Diamant for the statistical analysis, Dr M. Hofman for critically reading the manuscript, and Mr G. Van der Meulen for the photography.
Received for publication December 3, 2001; accepted December 13, 2001.
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