ARTICLE |
Correspondence to: Jean-Marc Fritschy, Inst. of Pharmacology, Univ. of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland..
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Summary |
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We designed a protocol to improve the immunohistochemical analysis of human brain structures, which overcomes the limited detection sensitivity, high background, and intense autofluorescence commonly associated with human tissue. This procedure was evaluated by using antibodies against major GABAA receptor subunits (1,
2,
3,
2) in autopsy and surgical specimens. Tissue blocks were briefly fixed by immersion and pretreated with microwave irradiation in sodium citrate buffer. Immunoperoxidase staining revealed a marked enhancement of cell surface immunoreactivity and reduction of background in microwave-irradiated tissue, irrespective of its origin. For confocal laser scanning microscopy, immunofluorescence staining was optimized with the tyramide signal amplification (TSA) technique. This procedure not only dramatically increased the sensitivity for antigen detection but also totally suppressed autofluorescence, thus revealing the cellular and subcellular distribution of GABAA receptor subunits. A distinct neuron-specific expression pattern of the
-subunit variants was observed in cerebral cortex and hippocampal formation, along with widespread expression of the
2-subunit. Of particular interest was the prominent
2- and
3-subunit staining on the initial axon segment of pyramidal neurons. This protocol represents a major improvement for high-resolution studies of human brain tissue aimed at investigating morphological alterations underlying neurological diseases. (J Histochem Cytochem 46:11291139, 1998)
Key Words: antigen retrieval, immunohistochemistry, autofluorescence, biopsy, confocal laser scanning microscopy, GABAA receptors, human, microwave irradiation, postmortem, tyramide
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Introduction |
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Analysis of the cellular and subcellular distribution of ion channels, neurotransmitter receptors, and transporters is essential for unraveling their role in synaptic transmission in normal and diseased brain. Although most current knowledge is derived from animal models, a better understanding of the pathophysiology of neurological and psychiatric disorders will ultimately depend on studies of the human brain. Thus far, two major technical limitations have hampered immunohistochemical analyses of human brain. First, optimal fixation is difficult to achieve, resulting in poor tissue preservation, limited sensitivity for antigen detection, and high background signals. Second, the intense autofluorescence originating from lipofuscin pigments largely precludes the use of epifluorescence and confocal laser scanning microscopy (for review see
Immunohistochemical methods have been significantly improved with the development of two novel approaches for increasing the sensitivity of antigen detection. The first technique, antigen retrieval through microwave irradiation, was designed primarily for histopathological analysis of formalin-fixed, paraffin-embedded sections of human tissue (-aminobutyric acid (GABAA) receptors in rat brain tissue (
In the present study we have established a protocol based on these two procedures to investigate the cellular and subcellular localization of GABAA receptor subunits in the human brain. These experiments were conducted as part of a larger project aimed at determining whether alterations in the expression of GABAA receptors contribute to the pathophysiology of epilepsy (16, ß13,
13,
,
,
13) (for review
The protocol presented here involves microwave irradiation to enhance antigen detection in immersion-fixed human brain tissue obtained at autopsy or surgery. GABAA receptor subunits were then visualized by immunofluorescence staining in cerebral cortex and hippocampus using subunit-specific antibodies in combination with an optimized TSA technique. The results demonstrate a dramatic increase in sensitivity and a complete suppression of autofluorescence signals, allowing detailed high-resolution analysis of the subcellular distribution of major GABAA receptor subunits in both autopsy and surgical specimens.
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Materials and Methods |
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Tissue Collection
Brains obtained at autopsy were dissected into 7- to 12-mm-thick blocks from cerebral cortex or hippocampal formation. The subjects, three men, had died at 40, 69, and 73 years of age and had no known history of neurological or psychiatric disease. The interval between death and tissue processing ranged from 8 to 16 hr. Specimens of cerebral cortex or hippocampus were resected from 15 patients with medically intractable frontal or temporal lobe epilepsy and collected immediately on resection. These procedures were performed in accordance with the Helsinki Declaration of 1975 and were approved by the Ethics Committee of the University Hospital Zurich. Tissue blocks were rinsed in PBS, pH 7.4, and immersion-fixed for 68 hr at 4C in a mixture of 4% freshly dissolved paraformaldehyde and 15% saturated picric acid in 0.15 M phosphate buffer, pH 7.4 (
Microwave Irradiation
Microwave pretreatment of the fixed tissue blocks was performed as described previously (
Immunohistochemistry
The antibodies tested included the monoclonal antibody bd-24 recognizing the human GABAA receptor 1-subunit (
2-,
3-, and
2-subunits. The preparation and characterization of the polyclonal antibodies, raised against synthetic peptides derived from rat cDNA sequences, have been described previously (see
Free-floating sections were processed for immunoperoxidase staining using the ABC method of 1-subunit (monoclonal antibody bd-24) 0.2 µg/ml;
2-subunit (affinity-purified) 1.3 µg/ml;
3-subunit (affinity-purified) 1.8 µg/ml; and
2-subunit (crude serum) 1:1500. Sections were then washed three times for 10 min with Tris-saline and incubated for 30 min with biotinylated secondary antibodies raised in goat (diluted 1:300 in Tris-saline containing 2% normal goat serum and 0.05% Triton X-100). After washing three times for 10 min with Tris-saline, sections were reacted with the ABC complex for 30 min (Vectastain Elite kit; Vector Laboratories, Burlingame, CA), washed again three times for 10 min, and finally incubated with 0.05% diaminobenzidine tetrahydrochloride (Sigma; St Louis, MO) and 0.001% hydrogen peroxide diluted in Tris-saline at pH 7.7. The staining reaction was carried out at RT for 515 min and was stopped by transferring the sections into ice-cold PBS. Sections were then washed three times for 10 min with PBS, mounted on gelatin-coated slides, air-dried, dehydrated with an ascending series of ethanol, and coverslipped out of xylene. Control experiments for staining specificity included replacement of primary antibodies with nonimmune serum and preabsoption of the antibodies with 35 µg/ml of their respective peptide antigen (
Immunofluorescence staining was performed using the protocol described in 2-subunit 2.5 µg/ml;
3-subunit 3 µg/ml; and
2-subunit 1:1000). They were then incubated with affinity-purified goat secondary antibodies (Jackson Immunoresearch; West Grove, PA) cou-pled to dichlorotriazinylaminofluorescein (DTAF; 1:100), carbocyanine (Cy2; 1:100), or indocarbocyanine (Cy3; 1: 300).
Immunofluorescence Staining with Tyramide Signal Amplification
The TSA kit used for the amplification procedure was obtained from NEN Life Science Products (Brüssels, Belgium). Free-floating sections were incubated overnight at 4C with primary antibodies diluted in 50 mM Tris-saline at pH 7.4 containing 4% normal goat serum and 0.05% Triton X-100. Several concentrations were tested, and the optimal dilutions of antibodies were as follows: 1-subunit (monoclonal antibody bd-24) 0.04 µg/ml;
2-subunit, 0.15 µg/ml;
3-subunit 0.09 µg/ml; and
2-subunit 1:10,000. Subsequent steps were performed at RT. Sections were washed three times for 10 min with Tris-saline and incubated for 60 min with biotinylated secondary antibodies raised in goat (diluted 1:500 in Tris-saline containing 4% normal goat serum and 0.05% Triton X-100). After washing three times for 10 min in TNT buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween-20), sections were blocked with TNB buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% NEN Blocking Reagent) for 30 min. They were then incubated for 30 min with streptavidinHRP diluted at 1:2000 in TNB. After washing three times for 10 min in TNT, the sections were incubated with biotinylated tyramide in amplification diluent. Several concentrations (1:50, 1:75, 1:150) and incubation times (3 min, 7 min, 10 min) were tested. The best results were obtained with a biotinylated tyramide dilution of 1:75 and an incubation time of 10 min. Thereafter, sections were rinsed three times for 10 min in TNT and then TNB. They were then incubated for 30 min in streptavidin conjugated either to Cy2 or Cy3 (diluted 1:1000 in TNB). Finally, sections were washed three times for 10 min with PBS, mounted on gelatin-coated slides, air-dried, and coverslipped with buffered glycerol.
Data Analysis and Photography
Sections were analyzed with a Zeiss Axiophot microscope (Jena, Germany) equipped for brightfield and epifluorescence microscopy. Photomicrographs were taken with Kodak T-max 100 film (Rochester, NY). The same sections were also analyzed by confocal laser scanning microscopy (TCS 4D; Leica, Heidelberg, Germany) using Imaris software (Bitplane; Zurich, Switzerland) for image processing. Digital images were printed with a Fuji Pictrography 3000 digital image printer (Tokyo, Japan).
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Results |
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Antigen Retrieval by Microwave Irradiation
The influence of microwave irradiation on GABAA receptor subunit immunoreactivity (IR) was evaluated in autopsy and surgical specimens of cerebral cortex or hippocampus using the monoclonal antibody bd-24, which recognizes the 1-subunit (
2-,
3-, and
2-subunits. With the standard immunoperoxidase protocol, bd-24 yielded a highly specific and differentiated staining pattern for the
1-subunit, as described previously in the human hippocampal formation (
3-subunit in frontal cortex. Microwave irradiation resulted in a marked increase of the signal-to-noise ratio by enhancing cell surface staining and decreasing nonspecific background staining. Thus, in microwave-irradiated tissue, the
3-subunit IR was found to label pyramidal neurons located in the deeper layers of the frontal cortex, revealing their dendrites extending towards superficial layers (Figure 1B). Whereas this pattern was largely conserved in frontal and entorhinal cortex, clear differences in staining intensity became apparent in the laminar distribution of
3-subunit IR. In frontal cortex there was intense and diffuse staining of the neuropil in Layer II and the superficial portion of Layer III, with more moderate staining in Layers V and VI (Figure 1B). In contrast, in the entorhinal cortex staining was intense in the deeper layers and light to moderate in Layers II and III. In addition, the cell islands of Layer II were devoid of
3-subunit IR (Figure 1C and Figure 1D).
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Pretreatment of fixed tissue with microwave irradiation proved to be equally effective for tissue obtained both at autopsy and at surgery, indicating that a postmortem interval of up to 16 hr did not significantly reduce GABAA receptor subunit IR. This is shown in the entorhinal cortex by comparing the GABAA receptor 3-subunit IR in tissue resected from a patient with intractable temporal lobe epilepsy (Figure 1C) and in control tissue obtained at autopsy (Figure 1D). In the surgical specimen, staining in layers II and III of the entorhinal cortex was slightly decreased compared with the autopsy sample (Figure 1C). This finding is most likely associated with the underlying chronic seizure disorder and may reflect neuronal cell loss in Layers II and III of the entorhinal cortex (
3-subunit IR was abundant, with a characteristic laminar distribution, although the patient had suffered a prolonged agonal state with progressive renal failure and the postmortem interval was 8 hr (Figure 1D). Furthermore, when the same region was compared in the three specimens obtained at autopsy, no significant difference in the intensity or specificity of GABAA receptor subunit staining was detected. For tissue obtained both at autopsy and at surgery, the best results were obtained when the 712-mm-thick tissue blocks were fixed for less than 8 hr and the irradiation time did not exceed 150 sec.
Increased Sensitivity and Elimination of Autofluorescence by TSA
For immunofluorescence staining, the microwave irradiation procedure alone was found to be inadequate because the strong autofluorescence occluded the weak GABAA receptor subunit IR. This is illustrated for the 2-subunit in the CA2 region (Figure 2A) and in the dentate gyrus and hilus of the hippocampus (Figure 2D). The TSA procedure was then tested in sections pretreated with microwave irradiation. This resulted in a striking increase in immunofluorescence staining intensity for the four GABAA receptor subunits investigated and in the elimination of autofluorescence, as illustrated in Figure 2C and Figure 2E. In the hippocampus, the pyramidal cell layer and the dendritic layers, as well as the interneurons, appeared intensely stained in sections processed with TSA, as shown for the
2-subunit (Figure 2C). Prominent staining of this subunit was also seen in the dentate gyrus, with the somata of granule cells being clearly outlined and the molecular layer strongly labeled (Figure 2E). In addition, cell somata in the hilus of the hippocampus were intensely stained, together with their long processes (Figure 2E). A comparable improvement in the detection of GABAA receptor subunits was observed for the
2 and
3 antisera and for bd-24 recognizing the
1-subunit. The distribution of the
2-subunit IR was similar to that of the
2-subunit, except that it was not detected in interneurons. The
3-subunit IR was particularly abundant in the CA1 region of the hippocampus, whereas the
1-subunit IR was most prominent in many interneurons distributed throughout the hippocampal formation. For all antibodies used, TSA led to a dramatic increase in sensitivity. For example, a 1:10,000 dilution of the
2 antiserum yielded intense staining, whereas the signals were barely detectable with a 1:1000 dilution in conventional immunofluorescence staining.
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The second major effect of TSA was suppression of autofluorescence, which was directly correlated with the duration of incubation with biotinylated tyramide. Although autofluorescence signals were reduced but still present after 3 min of incubation (Figure 2B), they had disappeared after 10 min of incubation with biotinylated tyramide (Figure 2C). The enhanced detection of GABAA receptor subunits and the elimination of autofluorescence were equally effective, regardless of the age at which the subjects had died or undergone surgery or of the premortem and postmortem conditions. Even in the oldest subjects (69 and 73 years of age) with the highest amount of lipofuscin granules, autofluorescence was totally suppressed.
Analysis of the Subcellular Distribution of GABAA Receptor Subunits
The strong immunofluorescent signals and the elimination of autofluorescence produced with TSA enabled us to investigate GABAA receptor subunit staining at the subcellular level with confocal laser scanning microscopy. Individual neurons were outlined along their surface by many puncta, most likely representing synaptic receptors, as shown for the 2-subunit in CA1 pyramidal neurons (Figure 3A) and for the
2-subunit in granule cells of the dentate gyrus (Figure 3B). Particularly strong staining of the
1-subunit was observed in mossy cells (Figure 3C), revealing the characteristic "clusters of spheres" that form the excrescences of this hilar cell type described by
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The most striking observation, however, was the intense 2-subunit IR on the axon initial segment (AIS) of pyramidal neurons in the hippocampus (Figure 3DF) and cerebral cortex (not shown). In contrast, the
3-subunit IR was mostly localized on the AIS of neocortical pyramidal neurons. The AIS of principal cells receives prominent GABAergic input from chandelier cells (
2-subunit IR revealed the AIS as forming 2040-µm-long tail-like structures outlined by many intensely stained hot spots as shown for the AIS of CA3 pyramidal neurons (Figure 3E and Figure 3F). Typically, the staining started at some distance (1020 µm) from the base of the soma. These features are similar to those reported for the rat, indicating that the subcellular distribution of the GABAA receptor
2-subunit in pyramidal neurons is largely conserved across species.
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Discussion |
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The protocol described in this report combines microwave pretreatment of fixed tissue and the TSA technique to overcome two major obstacles associated with immunohistochemical studies of neurotransmitter receptors in human brain tissue. Microwave irradiation significantly increases the signal-to-noise ratio by enhancing cell surface IR and reducing nonspecific staining, whereas TSA results in a striking boost in sensitivity for antigen detection and eliminates autofluorescence originating from lipofuscin pigments. Our preliminary analysis using this protocol reveals a differential and neuron-specific expression pattern of the major GABAA receptor subunits in cerebral cortex and hippocampal formation at both the regional and the cellular level.
Effects of Microwave Irradiation
Antigen retrieval by microwave irradiation has been reported to be effective for a large variety of antigens masked in paraffin-embedded and vibratome sections of formalin-fixed archival tissue (for reviews, see
The enhancement of cell surface IR observed for GABAA receptor subunits suggests that microwave irradiation facilitates the access of antibodies to their antigen, notably within synaptic clefts (
Microwave irradiation was equally effective for both autopsy and surgical specimens, indicating that GABAA receptor subunit antigens are stable for several hours after death. Furthermore, the quality of staining is comparable to that reported for perfusion-fixed rat brain tissue (
Effects of TSA on Immunofluorescence Staining
As a result of the remarkable increase in sensitivity induced by TSA, low concentrations of primary antibodies are sufficient for strong immunofluorescence staining. This represents a clear advantage, considering that polyclonal antisera may contribute substantially to background staining (
Analysis of the Distribution of GABAA Receptor Subunits
To date, high-affinity monoclonal antibodies such as bd-24 and bd-17 (recognizing the 1- and the ß2/ß3- subunits, respectively) have provided the best results for immunohistochemical visualization of GABAA receptors in the human brain (
-subunit variants (
2,
3,
5) because they represent receptor populations with distinct pharmacological profiles and with a characteristic distribution pattern in rodent brain (
2-subunit distribution is of particular relevance, considering that this subunit, which is found in approximately 95% of GABAA receptors in rat brain (
2- and
3-subunit IR in human brain. A number of similarities were observed in the expression pattern of these subunits compared to the rat. For example, in both rat and human, the
3-subunit predominates in deep cortical layers and is found in dendrites extending towards the pial surface, whereas the
2-subunit is very abundant throughout the hippocampal formation in pyramidal cells and dentate gyrus granule cells. The similarity extends to the subcellular distribution of receptor subtypes, with the
2-subunit IR being enriched in the AIS of hippocampal and neocortical pyramidal cells. However, clear differences were also observed. For example, the
3-subunit is particularly intense in the CA1 region of the human hippocampus whereas it is virtually absent in the rat (
The detection of presumptive synaptic GABAA receptors in human brain, as revealed by the intense punctate staining outlining cell bodies and dendrites and by the prominent 2- and
3-subunit IR on AIS of pyramidal cells, demonstrates both the sensitivity achieved with our protocol and the fact that these proteins are stable enough to withstand postmortem delays and tissue processing. A dysfunction in the GABAergic transmitter system is believed to contribute to the pathophysiology of epilepsy (for review see
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Acknowledgments |
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Supported by the Théodore OTT Fund.
We are grateful to Dr Hanns Möhler for his continuous support and we thank Drs Thomas Bächi and Matthias Höchli for their competent help with confocal laser scanning microscopy.
Received for publication March 13, 1998; accepted June 16, 1998.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams JC (1992) Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem 40:1457-1463
Ainley CD, Ironside JW (1994) Microwave technology in diagnostic neuropathology. J Neurosci Methods 55:183-190[Medline]
Amaral DG (1978) A Golgi study of cell types in the hilar region of the hippocampus in the rat. J Comp Neurol 182:851-914[Medline]
Belichenko PV, Fedorov AA, Dahlstrom AB (1996) Quantitative analysis of immunofluorescence and lipofuscin distribution in human cortical areas by dual-channel confocal laser scanning microscopy. J Neurosci Methods 69:155-161[Medline]
Benke D, Honer M, Michel C, Mohler H (1996) GABAA receptor subtypes differentiated by their -subunit variants: prevalence, pharmacology and subunit architecture. Neuropharmacology 35:1413-1423[Medline]
Berghorn KA, Bonnett JH, Hoffman GE (1994) cFos immunoreactivity is enhanced with biotin amplification. J Histochem Cytochem 42:1635-1642
Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 125:279-285[Medline]
Bobrow MN, Shaughnessy KJ, Litt GJ (1991) Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays. J Immunol Methods 137:103-112[Medline]
Bohlhalter S, Weinmann O, Mohler H, Fritschy JM (1996) Laminar compartmentalization of GABAA-receptor subtypes in the spinal cord: an immunohistochemical study. J Neurosci 16:283-297[Abstract]
De Haas RR, Verwoerd NP, van der Corput MP, van Gijlswijk RP, Siitari H, Tanke HJ (1996) The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. J Histochem Cytochem 44:1091-1099
Du F, Whetsell WO, Jr, Abou-Khalil B, Blumenkopf B, Lothman EW, Schwarcz R (1993) Preferential neuronal loss in layer III of the entorhinal cortex in patients with temporal lobe epilepsy. Epilepsy Res 16:223-233[Medline]
Evers P, Uylings HBM (1997) An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. J Neurosci Methods 72:197-207[Medline]
Evers P, Uylings HBM (1994a) Effects of microwave pretreatment on immunocytochemical staining of vibratome sections and tissue blocks of human cerebral cortex stored in formaldehyde fixative for long periods. J Neurosci Methods 55:163-172[Medline]
Evers P, Uylings HBM (1994b) Microwave-stimulated antigen retrieval is pH and temperature dependent. J Histochem Cytochem 42:1555-1563
Ewert M, Shivers BD, Luddens H, Mohler H, Seeburg PH (1990) Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor. J Cell Biol 110:2043-2048[Abstract]
Faull RL, Waldvogel HJ, Nicholson LFB, Synek BJL (1993) The distribution of GABAA-benzodiazepine receptors in the basal ganglia in Huntington's disease and in the quinolinic acid-lesioned rat. Prog Brain Res 99:105-123[Medline]
Fox CH, Johnson FB, Whiting J, Roller PP (1985) Formaldehyde fixation. J Histochem Cytochem 33:845-853[Medline]
Fritschy JM, Mohler H (1995) GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J Comp Neurol 359:154-194[Medline]
Fritschy JM, Weinmann O, Wenzel A, Benke D (1998) Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry. J Comp Neurol 390:194-210[Medline]
Gao B, Fritschy JM (1994) Selective allocation of GABAA receptors containing the 1 subunit to neurochemically distinct subpopulations of rat hippocampal interneurons. Eur J Neurosci 6:837-853[Medline]
Gao B, Hornung JP, Fritschy JM (1995) Identification of distinct GABAA-receptor subtypes in cholinergic and parvalbumin-positive neurons of the rat and marmoset medial septum-diagonal band complex. Neuroscience 65:101-117[Medline]
Harman D (1989) Lipofuscin and ceroid formation: the cellular recycling system. Adv Exp Med Biol 266:3-15[Medline]
Hendry SHC, Huntsman MM, Vinuela A, Mohler H, de Blas AL, Jones EG (1994) GABAA receptor subunit immunoreactivity in primate visual cortex: distribution in macaques and humans and regulation by visual input in adulthood. J Neurosci 14:2383-2401[Abstract]
Houser CR, Olsen RW, Richards JG, Mohler H (1988) Immunohistochemical localization of benzodiazepine/GABAA receptors in the human hippocampal formation. J Neurosci 8:1370-1383[Abstract]
Hsu SM, Raine L, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580[Abstract]
Kerstens HMJ, Poddighe PJ, Hanselaar AGJM (1995) A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J Histochem Cytochem 43:347-352
Kosaka T (1980) The axon initial segment as a synaptic site: ultrastructure and synaptology of the initial segment of the pyramidal cell in the rat hippocampus (CA3 region). J Neurocytol 9:861-882[Medline]
Loup F, Wieser HG, Yonekawa Y, Mohler H, Fritschy JM (1997) Subtype-specific upregulation of GABAA-receptors in the hippocampal formation of temporal lobe epilepsy patients. Soc Neurosci Abstr 23:816
Macdonald RL, Olsen RW (1994) GABAA receptor channels. Annu Rev Neurosci 17:569-602[Medline]
Macechko PT, Krueger L, Hirsch B, Erlandsen SL (1997) Comparison of immunologic amplification vs enzymatic deposition of fluorochrome-conjugated tyramide as detection systems for FISH. J Histochem Cytochem 45:359-363
Marriott T, Clegg RM, ArndtJovin DJ, Jovin TM (1991) Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging. Biophys J 60:1374-1387[Abstract]
Mayer G, Bendayan M (1997) Biotinyl-tyramide: a novel approach for electron microscopic immunocytochemistry. J Histochem Cytochem 45:1449-1454
McKernan RM, Whiting PJ (1996) Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 19:139-143[Medline]
Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orscheschek K, Moubayed P, Feller AC (1995) Immunomax. A maximized immunohistochemical method for the retrieval and enhancement of hidden antigens. Lab Invest 73:149-156[Medline]
Mohler H, Benke D, Benson J, Luscher B, Rudolph U, Fritschy JM (1997) Diversity in structure, pharmacology, and regulation of GABAA receptors. In Enna SJ, Bowery NG, eds. The GABA Receptors. Totowa, NJ, Humana Press, 11-36
Munakata S, Hendricks JB (1993) Effect of fixation time and microwave oven heating time on retrieval of the Ki-67 antigen from paraffin-embedded tissue. J Histochem Cytochem 41:1241-1246
Nusser Z, Sieghart W, Benke D, Fritschy JM, Somogyi P (1996) Differential synaptic localization of two major -aminobutyric acid type A receptor
subunits on hippocampal pyramidal cells. Proc Natl Acad Sci USA 93:11939-11944
Olsen RW, Avoli M (1997) GABA and epileptogenesis. Epilepsia 38:399-407[Medline]
Raap AK, van der Korput MP, Vervenne RA, van Gijlswijk RPM, Tanke HJ, Wiegant J (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet 4:529-534[Abstract]
Schmidt BF, Chao J, Zhu Z, DeBiasio RL, Fisher G (1997) Signal amplification in the detection of single-copy DNA and RNA by enzyme-catalyzed deposition (CARD) of the novel fluorescent reporter substrate Cy3.29-tyramide. J Histochem Cytochem 45:365-373
Schoch P, Richards JG, Haring P, Takacs B, Stahli C, Staehelin T, Haefely W, Mohler H (1985) Co-localization of GABAA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies. Nature 314:168-171[Medline]
Seveus L, Vaisala M, Syrjanen S, Sandberg M, Kuusisto A, Harju R, Salo J, Hemmila I, Kojola H, Soini E (1992) Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization. Cytometry 13:329-338[Medline]
Shi SR, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem 45:327-343
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741-748[Abstract]
Shindler KS, Roth KA (1996) Double immunofluorescent staining using two unconjugated primary antisera raised in the same species. J Histochem Cytochem 44:1331-1335
Sieghart W (1995) Structure and pharmacology of -aminobutyric acidA receptor subtypes. Pharmacol Rev 47:181-234[Medline]
Somogyi P, Fritschy JM, Benke D, Roberts JDB, Sieghart W (1996) The 2 subunit of the GABAA receptor is concentrated in synaptic junctions containing the
1 and ß2/3 subunits in hippocampus, cerebellum and globus pallidus. Neuropharmacology 35:1425-1444[Medline]
Somogyi P, Nunzi MG, Gorio A, Smith AD (1983) A new type of specific interneuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells. Brain Res 259:137-142[Medline]
Somogyi P, Takagi H (1982) A note on the use of picric acid-paraformaldehyde-glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry. Neuroscience 7:1779-1783[Medline]
Speel EJM, Ramaekers FCS, Hopman AHN (1997) Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification. J Histochem Cytochem 45:1439-1446
Strappe PM, Wang TH, McKenzie CA, Lowrie S, Simmonds P, Bell JE (1997) Enhancement of immunohistochemical detection of HIV-1 p24 antigen in brain by tyramide signal amplification. J Virol Methods 67:103-112[Medline]
Van de Lest CHA, Versteeg EMM, Veerkamp JH, van Kuppevelt TH (1995) Elimination of autofluorescence in immunofluorescence microscopy with digital image processing. J Histochem Cytochem 43:727-730
Van Gijlswijk RPM, Zijlmans HJMAA, Wiegant J, Bobrow MN, Erickson TJ, Adler KE, Tanke HJ, Raap AK (1997) Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem 45:375-382
Van heusden J, de Jong P, Ramaekers F, Bruwiere H, Borgers M, Smets G (1997) Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels. J Histochem Cytochem 45:315-319
von Wasielewski R, Mengel M, Gignac S, Wilkens L, Werner M, Georgii A (1997) Tyramine amplification technique in routine immunohistochemistry. J Histochem Cytochem 45:1455-1459
Werner M, von Wasielewski R, Komminoth P (1996) Antigen retrieval, signal amplification and intensification in immunohistochemistry. Histochem Cell Biol 105:253-260[Medline]
Yachnis AT, Trojanowski JQ (1994) Studies of childhood brain tumors using immunohistochemistry and microwave technology: methodological considerations. J Neurosci Methods 55:191-200[Medline]