ARTICLE |
Correspondence to: Arno Schad, Inst. for Anatomy and Cell Biology, Dept. of Medical Cell Biology, University of Heidelberg, INF 307, D-69120 Heidelberg, Germany. E-mail: schad@medicusnet.de
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Summary |
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Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging. (J Histochem Cytochem 51:751760, 2003)
Key Words: catalase, brain, in situ hybridization, mRNA, CARD, digoxigenin, peroxisome, GAPDH, immunohistochemistry, immunofluorescence, confocal laser scanning, microscopy
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Introduction |
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CATALASE is the marker enzyme for peroxisomes and decomposes the H2O2 produced by the peroxisomal oxidases (
Whereas cultured neurons and astroglial cells contain both catalase and the glutathione system for peroxide detoxification (
Catalase has been visualized by the alkaline diaminobenzidine (DAB) reaction (
The mRNA encoding for catalase has been demonstrated by Northern blotting in different rat brain regions (
We recently described a nonradioactive ISH protocol for detection of mRNAs encoding peroxisomal matrix and membrane proteins (
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Materials and Methods |
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Tissue Preparation
Adult male SpragueDawley rats weighing 220250 g were perfused under chloral hydrate anesthesia via the abdominal aorta. After a 30-sec rinse with physiological saline, a fixative containing 0.25% glutaraldehyde, 2% sucrose in 0.1M PIPES buffer (pH 7.4) was perfused for 5 min (for the IHC experiments, 4% freshly prepared paraformaldehyde in the same buffer was used instead of glutaraldehyde). The brains were cut into 12-mm frontal or sagittal slices and were embedded in paraffin using an automated vacuum tissue processor (Shandon; Frankfurt, Germany). For experiments, 4-µm sections were cut and mounted on Super Frost Plus slides (Menzel; Braunschweig, Germany).
Preparation of Digoxigenin-labeled Riboprobes
Digoxigenin-labeled riboprobes were produced by in vitro transcription of specific DNA fragments obtained by PCR amplification of plasmid vectors containing the cDNA for rat catalase in a pGEM 7Zf () vector and GAPDH in a pBluescript II vector (
In vitro transcription was performed using a digoxigenin labeling kit from Roche Diagnostics (Mannheim, Germany) and the resulting sense and antisense transcripts were cleaved to an average length of 200 bases by alkaline hydrolysis before their use in the ISH experiments.
In Situ Hybridization
Dewaxed and rehydrated paraffin sections were subjected to prehybridization steps including deproteination with 0.1 M HCl for 10 min followed by two PBS (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4) rinses for 5 min each. Proteinase K digestion was carried out at 37C for 30 min at a concentration of 1030 µg/ml in a TE buffer (100 mM Tris, 50 mM EDTA, pH 8.0), followed by postfixation in 4% freshly depolymerized paraformaldehyde in PBS. After two PBS rinses the sections were acetylated for 20 min with 0.25% (v|v) acetic anhydride in a TEA buffer (0.1 M triethanolamine, pH 8.0), rinsed in PBS, dehydrated, and air-dried. Before the hybridization the sections were prehybridized for 2 hr at 45C with a mixture consisting of 50% formamide, 50 mM Tris, 25 mM EDTA, 20 mM NaCl, 250 µg/ml yeast tRNA, and 2.5 x Denhardt's solution. Hybridization was carried out overnight at 45C with 5 ng/µl riboprobe in a solution containing 50% formamide, 20 mM Tris, 1 mM EDTA, 0.33 M NaCl, 0.5 µg/µl tRNA, 0.1 µg/µl poly-A RNA, 10% dextran sulfate, and 1 x Denhardt's solution. For each section, 20 µl of this hybridization mix, covered with "Hybri-slips" (Sigma; Deisenhofen, Germany), was used.
The posthybridization washes included a 30-min rinse at 53C in 2 x SSC, followed by 1 hr at 53C with 1 x SSC and 50% formamide. Then the sections were passed through two changes of 0.5 x SSC and 0.2 x SSC for 10 min each at room temperature. After a 10-min rinse in TBS (150 mM NaCl, 100 mM Tris, pH 7.5), nonspecific protein-binding sites were blocked by a 30-min incubation with a blocking buffer containing 1% (w|v) blocking reagent (Roche Diagnostics) and 0.5% (w|v) BSA in TBS buffer.
For detection of the hybridized probe, the sections were incubated overnight with peroxidase-labeled Fab fragments directed against digoxigenin diluted 1:100 in TNB buffer [0.5% (w|v) blocking medium (NEN-Life Science; Cologne, Germany) in TBS]. After two 5-min washing steps in TNT buffer (0.05% Tween-20 in TBS), a 30-min period in TNB buffer, and another two 5-min changes in TNT buffer, the biotinylated tyramine was applied to the sections as a 1:50 dilution in amplificant diluent (NEN-Life Science) for 20 min. After three 5-min changes of TNT buffer the sections were incubated with a 1:100 dilution of streptavidinHRP conjugates in TNB buffer for 30 min. The color reaction for visualization of the peroxidase activity was performed after three 5-min rinses with TNT buffer using AEC as the substrate for peroxidase for 5 min. Afterwards the sections were rinsed in distilled water, counterstained with hematoxylin, and mounted with glycerolgelatin. Negative controls were prepared using the corresponding sense probes.
Some parallel sections, after hybridization and posthybridization as described, were incubated with anti-digoxigenin Fab fragments conjugated with alkaline phosphatase instead of peroxidase to obtain a direct NBT/BCIP color reaction without amplification, as described previously (
Immunohistochemistry
IHC for detection of catalase was performed on paraffin sections of rat brain samples fixed by perfusion with a fixative containing 4% freshly prepared paraformaldehyde in 0.1 M PIPES buffer (pH 7.4). For antigen retrieval, the protocol recently described by
Double-labeling Immunofluorescence
For simultaneous detection of catalase antigen with the glial marker GFAP for astrocytes or CNPase for oligodendrocytes, additional double-labeling immunofluorescence experiments were performed. At first the paraformaldehyde-fixed, paraffin-embedded sections were processed for catalase detection as described above. Then the anti-catalase antibody was detected with an Alexa-488-labeled goat anti-rabbit antibody (Molecular Probes, Eugene, OR; diluted 1:350). Then the sections were incubated overnight with mouse monoclonal antibodies against GFAP (Sigma, St Louis, MO; clone G-A-5, diluted 1:1000) or CNPase (Abcam, Cambridge, UK; clone ab3619, diluted 1:1000). The monoclonal antibodies were detected with an Alexa-594-labeled donkey anti-mouse antibody (Molecular Probes; diluted 1:500). After nuclear counterstaining with TO-PRO-3 (Molecular Probes), the sections were mounted in Mowiol 4-88 (Hoechst; Frankfurt/M., Germany) with 0.5% N-propylgallate.
The sections were analyzed using a Leica TCS SP confocal microscope (Leica Microsystems; Heidelberg, Germany) with sequential detection of the Alexa-488 fluorescence at 497643 nm (excited at 488 nm, green), the Alexa-594 emission at 600640 nm (excited at 544 nm, red), and of the TO-PRO-3 emission at 640761 nm (excited at 633 nm, printed in blue).
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Results |
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Positive staining representing the catalase mRNA was seen in the cytoplasm of neurons at many sites in rat brain with various intensities. In rat hippocampus, the mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as positive control, showed a strong neuronal expression pattern, with prominent cytoplasmic staining and negative nuclei and nucleoli (Fig 1a and Fig 1c). The intensity of staining could not be further enhanced by prolongation of the incubation time. The catalase mRNA, in comparison to GAPDH, showed a somewhat weaker expression, which could be demonstrated only by the use of CARD as a clear cytoplasmic staining (Fig 1b and Fig 1d). In the hippocampus, pyramidal cells were positive for catalase mRNA and, at lower signal intensity, the neurons of the dentate gyrus (Fig 1b). The frequency of strongly positive neurons was particularly high in the brainstem, where neurons of different cranial nerve nuclei, such as the trigeminal motor nucleus, showed strong catalase mRNA expression (Fig 1f). In cerebellum, Purkinje cells were distinctly positive (Fig 2a), with negative sense controls (Fig 2b). The olfactory bulb, together with brainstem and cerebellum, belongs to the regions of rat brain with the highest catalase mRNA expression, showing a strong staining in the mitral cells and of neurons of the stratum plexiforme externum (Fig 2c). The corresponding sense controls were again completely negative (Fig 2d).
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In general, the detection of catalase mRNA, in spite of CARD amplification, appeared to be restricted mostly to neurons. The many oligodendrocytes of the corpus callosum, the perivascular astrocytes, and the endothelial cells remained negative. Nevertheless, the staining of catalase mRNA in neurons (e.g., in locus coeruleus; Fig 2e) was often accompanied by positive reaction in smaller cells which, without specific double labeling, could not be definitely ruled out to be of glial origin (arrows in Fig 2e).
To assess the role of signal amplification by CARD, some sections were hybridized with the same protocol but stained instead with alkaline phosphatase-labeled anti-digoxigenin Fab fragments and NBT/BCIP substrate (
To assess the validity of our improved ISH protocol, we compared the distribution of catalase mRNA with that of catalase protein using similarly processed paraffin-embedded sections and antigen retrieval (
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Because of the paucity of catalase mRNA signal in non-neuronal cells (see above), we assessed the presence of catalase immunostaining by using CLSM and double immunofluorescence. Antibodies to GFAP and CNPase were applied as specific markers for astroglial cells and oligodendrocytes, respectively. Finely granular immunofluorescence was observed most prominently as expected in neurons (Fig 4a and Fig 4b) and also in astroglial cells (Fig 4a4d) and in oligodendrocytes (Fig 4e and Fig 4f). The few catalase-positive granules in the latter cells were mostly localized in the perinuclear cytoplasm, particularly in oligodendrocytes (Fig 4e and Fig 4f).
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Discussion |
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Distribution of Catalase mRNA by ISH Corresponds to That of Catalase Protein
The present study has revealed the presence of catalase mRNA in the cytoplasm of neuronal cells in rat brain, corresponding to the distribution of catalase protein as reported previously by IHC (
Signal Amplification by CARD
The introduction of tyramine signal amplification has significantly improved the sensitivity of the peroxidase-based color reactions for IHC and ISH techniques. In this study, CARD was applied to enhance the sensitivity of a nonradioactive ISH protocol (
The high sensitivity of our ISH protocol allowed the use of glutaraldehyde for the perfusion fixation which, compared to paraformaldehyde, led to better tissue preservation. The improved quality of the morphological preparations combined with the high sensitivity of the detection protocol substantially exceeded the resolution of the only previously reported radioactive ISH protocol for catalase in brain (
The Important Role of Catalase and Peroxisomes in Brain
The localization of catalase mRNA by ISH revealed a heterogeneously distributed expression pattern along different subsets of neurons in rat brain. Not only monoaminergic neurons, such as those of the locus ceruleus, but also the GABAergic Purkinje cells, the glutamate-containing mitral cells of the olfactory bulb, and the hippocampal pyramidal cells appear to contain substantial amounts of catalase mRNA. As mentioned above, this correlates well with the immunocytochemical data on distribution of catalase protein in rat brain (
As a marker protein for peroxisomes, catalase is subject to severe alterations not only in primary genetic peroxisomal disorders, such as Zellweger syndrome, adrenoleukodystrophy, and infantile Refsum's disease (
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Acknowledgments |
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Supported by grants Ba 1155/14, Fa 146/13, and SFB 601 from the Deutsche Forschungsgemeinschaft and by a fellowship from the Christine-Jung-Stiftung to A.S. by the Medical Faculty of the University of Heidelberg.
The technical assistance of Inge Frommer is gratefully acknowledged.
Received for publication January 25, 2002; accepted January 22, 2003.
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