ARTICLE |
Correspondence to: H. Dariush Fahimi, Dept. of Anatomy and Cell Biology, U. of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany. E-mail: H.Dariush.Fahimi@urz.uni-heidelberg.de
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Summary |
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We present a protocol for detection of peroxisomal proteins and their corresponding mRNAs on consecutive serial sections of fetal and newborn mouse tissues by immunohistochemistry (IHC) and nonradioactive in situ hybridization (ISH). The use of perfusionfixation with depolymerized paraformaldehyde combined with paraffin embedding and digoxigenin-labeled cRNA probes provided a highly sensitive ISH protocol, which also permitted immunodetection with high optical resolution by light and/or fluorescence microscopy. Signal enhancement was achieved by the addition of polyvinyl alcohol (PVA) for ISH color development. For IHC, signal amplification was obtained by antigen retrieval combined with biotinavidinHRP and Nova Red as substrate or by the catalyzed reporter deposition of fluorescent tyramide. Using this protocol, we studied the developmental changes in localization of the peroxisomal marker enzymes catalase (CAT) and acyl-CoA oxidase 1 (AOX), the key regulatory enzyme of peroxisomal ß-oxidation, at the protein and mRNA levels in mice from embryonic Day 14.5 to birth (P0.5). The mRNA signals for CAT and AOX were detected in sections of complete fetuses, revealing organ- and cell-specific variations. Here we focus on the localization patterns in liver, intestine, and skin, which showed increasing mRNA amounts during development, with the strongest signals in newborns (P0.5). Immunolocalization of the corresponding proteins revealed, in close correlation with the mRNAs, a distinct punctate staining pattern corresponding to the distribution of peroxisomes. (J Histochem Cytochem 49:155164, 2001)
Key Words: peroxisomes, mouse development, catalase, acyl-CoA oxidase, in situ hybridization, immunohistochemistry, fluorescence microscopy
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Introduction |
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Peroxisomes play an important role in lipid metabolism, with synthesis of etherlipids and cholesterol and degradation of very long-chain fatty acids, phytanic acid, and bioactive lipids such as prostaglandins and leukotrienes (
Although many peroxisomal proteins have been characterized at the molecular level in recent years, there are only a few reports on the distribution of their mRNA transcripts in different organs, mostly using radioactive ISH because of the relatively low expression levels (
Because of increasing interest in mouse development in conjunction with transgenic mouse models of human diseases, we decided to investigate the localization of peroxisomal proteins and their corresponding mRNAs in fetal and newborn mice by IHC and nonradioactive ISH. In this article we present a protocol to detect two such proteins and the corresponding mRNAs on consecutive sections of fetal and newborn mouse tissues. We demonstrate the presence of CAT and AOX mRNAs and proteins from 14.5 days p.c. (E14.5) to newborn mice (P0.5) in liver, intestine, and skin. CAT protein is detected in small cytoplasmic granules, resembling peroxisomes. An abstract with results of this study was presented at the Annual Meeting of the German Society for Cell Biology (
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Materials and Methods |
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Animals
Swiss mice were kept on a normal laboratory diet and water ad libitum. They were housed in cages in compliance with the guidelines for the humane care and use of laboratory animals of the Federal Republic of Germany. Female mice were placed with males overnight and were considered to be 0.5 days p.c. (post coitum) when a vaginal plug was present the following morning.
Fixation and Tissue Processing
Pregnant females of gestational stages E14.5, E15.5, E16.5, E17.5, and E18.5 days p.c. and newborns were anesthesized with diethylether. The fetuses delivered by C-section and newborns were perfused through the heart with 4% freshly depolymerized paraformaldehyde in PBS (10 mM phosphate buffer with 137 mM NaCl and 2.7 mM KCl at pH 7.4). After postfixation for 24 hr by immersion in the same fixative, the animals were sectioned sagittally in two halves and embedded in paraffin (Paraplast Plus; Sherwood Medical, St Louis, MO) using an automated vacuum infiltration tissue processor (Tissue-Tek V.I.P. E300; Sakura Finetek, Torrance, CA). Three-µm sections of whole animals were cut on a sliding microtome (Leica SM 2000 R; Leica Instruments, Nussloch, Germany) and mounted on Superfrost Plus slides (Shandon; Frankfurt/M., Germany).
In Situ Hybridization
After deparaffinization and rehydration, the sections were pretreated with 100 mM HCl and digested for 30 min at 37C with 520 µg/ml proteinase K (Sigma; Deisenhofen, Germany) in TE buffer (100 mM Tris, 50 mM EDTA, pH 8.0). The exact concentration of proteinase K was determined empirically for each block (
Synthesis of the digoxigenin-labeled riboprobes for CAT and AOX and their hydrolysis to 200-base fragments were as described previously (
Immunohistochemistry
For antigen retrieval and improved accessibility of epitopes, deparaffinized and rehydrated sections were subjected to any one of the following procedures: digestion with 0.01% trypsin (Sigma) in PBS for 10 min; digestion with 0.1% trypsin for 5 min; microwaving in 10 mM citrate buffer at pH 6.0 for 15 min at 720 W in a conventional household microwave oven (Panasonic), followed by 20 min cooling without changing buffer; or 0.01% trypsin for 5 min followed by 15 min of microwaving. Endogenous peroxidase was inhibited by 3% H2O2 for 5 min and nonspecific binding sites were blocked by 0.5% blocking reagent (NEN Life Science; Boston, MA) according to the manufacturer's recommendation. The endogenous biotin was blocked with an avidinbiotin blocking kit (Vector Laboratories; Burlingame, CA), and sections were incubated overnight at 4C with monospecific antibodies to CAT and AOX, which were characterized earlier (
Immunofluorescence
In addition to carbamazoles, the antigen binding sites were also detected by the use of fluorescein isothiocyanate (FITC)-labeled tyramides (NEN Life Science) according to the manufacturer's specifications. After counterstaining of nuclei with 4'-6-diamidino-2-phenylindole (DAPI), the slides were mounted in Mowiol 4-88 (Hoechst; Frankfurt/M., Germany) with 0.5% N-propylgallate to retard photobleaching.
Illustrations
The light microscopic sections were analyzed using a Leica DMLB microscope (Leica; Wetzlar, Germany). Digitized images were obtained with a color photo scanner (Kaiser; Buchen, Germany) and processed with Adobe Photoshop (version 5.5) on an Apple MacIntosh computer. Fluorescence preparations were examined using a Leica TCS MP confocal microscope (Leica Microsystems; Heidelberg, Germany). DAPI counterstain was excited with a Spectra Physics/Tsunami multi photon laser system (Spectra Physics; Mountain View, CA).
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Results |
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In Situ Hybridization
The mRNAs for CAT and AOX were expressed as early as gestational stage E14.5, increasing until birth and with the highest abundance in newborns. They were co-expressed within the same cells, with a higher signal intensity for CAT mRNA.
Clear ISH signal enhancement was achieved by the addition of 10% polyvinyl alcohol (70100 kD) to the alkaline phosphatase BCIP/NBT substrate, allowing detection of mRNAs of peroxisomal proteins also in tissues with low copy numbers (Fig 1a). Control incubations using mRNA (sense) probes for hybridization were consistently negative, confirming the specificity of the method (Fig 1b).
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Application of this improved protocol to sagittal sections of complete mouse fetuses revealed the presence of CAT and AOX mRNA in different organs (Fig 1a). Strong signals were obtained in liver, brain, kidney, brown adipose tissue, intestine, and skin. Moderate signals were also detected in thyroid, adrenals, heart, skeletal muscle, respiratory tract, renal tubules, and salivary glands, and weak signals in smooth muscle. Details on cellular and subcellular localization in selected organs (liver, intestine, and skin) are presented below, together with immunocytochemical data.
Immunohistochemistry
IHC analysis of sections incubated with antibodies against CAT showed strong staining with a weaker reaction for AOX in different tissues, corresponding to the localization of mRNAs in parallel sections by ISH (Fig 1c). The use of antigen retrieval by enzyme digestion or microwaving in citrate buffer resulted in strong signal enhancement, with further improvement by the combination of both, weak digestion (5 min with 0.01% trypsin) followed by microwaving (15 min) (Fig 2).
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At low magnification, the use of AEC as substrate for the final horseradish peroxidase enzyme reaction showed comparable results to Nova Red (data not shown), but at higher magnifications only the Nova Red substrate showed a punctate staining pattern (Fig 3), contrasting the diffuse cytoplasmic AEC staining (data not shown). The best subcellular resolution was achieved by the use of fluorescein-coupled tyramine as substrate for the final HRP reaction. Further improvements in resolution were obtained by the use of confocal laser scanning microscopy for analysis of sections (Fig 3i). In contrast, the use of directly fluorescent (DTAF-conjugated) secondary antibodies resulted in much lower fluorescence intensity (not shown), emphasizing the strong signal enhancement by the biotinstreptavidinHRP method combined with catalyzed reporter deposition (CARD) of fluorophore-labeled tyramides. In general, the granular staining pattern for peroxisomes was best obtained with the antibody to CAT, while the AOX antibody gave a rather diffuse cytoplasmic staining (data not shown). No immunoreactivity was found in parallel incubations of control sections when the primary antibody was omitted or replaced by preimmune serum throughout all combinations of secondary antibodies and signal enhancement methods (not shown).
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Liver
In the developing mouse liver, the mRNAs for peroxisomal proteins were mainly localized in the cytoplasm of hepatic parenchymal cells and were almost absent in normoblasts and megakaryocytes (Fig 1d). The absence of staining in nuclei and nucleoli and the complete absence of signal in control hybridizations using sense mRNA (Fig 1g) confirmed the specificity of the hybridization. By IHC, peroxisomes are detectable as early as gestational stage E14.5, where the few small hepatocytes were best recognized by the staining of peroxisomes (Fig 3a). In newborn mouse liver, the number and size of hepatocytes increased and they contained distinct granules corresponding to the distribution of peroxisomes (Fig 3b and Fig 3c).
Intestine
The mRNAs for CAT and AOX in the small intestine were mainly localized in enterocytes and, to a lesser extent, in developing smooth muscle cells (Fig 1e). Regarding the cryptvillous axis, the enterocytes showed similar staining intensity and no significant difference between the villi and crypts. In contrast to the homogeneous cytoplasmic distribution of mRNA, the peroxisomes of enterocytes, as detected by IHC, were mainly localized to the supranuclear area of the cytoplasm (Fig 2c), with an intensification of this phenomenon during development from gestational stage E14.5 to E18.5 (Fig 3d3f). At higher magnification, we observed a certain degree of heterogeneity in the intensity of CAT immunostaining in different peroxisomes even within the same cells (Fig 3f).
Skin
Fig 1f shows the distribution of CAT mRNA in the skin of a newborn mouse, which is mainly localized in suprabasal layers of epidermis and, to a lesser extent, in hair follicles. The corresponding sense control is negative (Fig 1i). The onset of catalase immunostaining, in contrast to liver and intestine, first appeared weak and diffuse (E14.5E16.5), and punctate staining of small peroxisomes was detected only at late pregnancy at E18.5 (Fig 3g), with no intensification of staining thereafter (Fig 3h and Fig 3i). Similar to the distribution of mRNA, peroxisomes were also more concentrated in the suprabasal layers of epidermis. Staining of hair follicles for CAT remained diffuse even at birth.
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Discussion |
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The results described here establish for the first time the histochemical and cytochemical localization of peroxisomal proteins and corresponding mRNAs in tissues of fetal and newborn mice at high subcellular resolution. Moreover, in serial sagittal sections of complete animals, we could show the distribution of mRNAs and the corresponding proteins. Whereas the mRNAs were exclusively in the cytoplasm, the proteins were found in fine granules corresponding to peroxisomes. This was made possible by combining our original protocol for ISH (
Fixation and Embedding
The method of perfusionfixation with aldehydes has been shown to be superior to immersionfixation, not only for ultrastructural preservation (
Antigen Retrieval
Paraformaldehyde fixation and paraffin embedding are known to reduce antigenicity caused by crosslinking of epitopes and changes of tertiary and quaternary protein structures (
Improved Detection of ISH and IHC Signals
In enzyme cytochemistry, it is well established that improved localization of enzyme activity can be achieved through the addition of viscosity-increasing polymers to the incubation media, which has also been adapted to visualize ISH signals (
In IHC, the new HRP substrate Nova Red was superior to the well-established AEC by yielding higher signal intensity and superior optical resolution of enzyme binding sites. Using the new substrate, peroxisomes could be detected by their punctate staining pattern (Fig 2 and Fig 3), whereas AEC as substrate resulted in a more diffuse staining in parallel sections. Because the Nova Red reaction product is insoluble in organic media, it was possible to dehydrate the sections and mount them with permanent mounting medium for prolonged storage.
To obtain better microscopic resolution for detection of immunostaining of peroxisomes, we also used fluorescence microscopy. Paraformaldehyde-fixed tissue is known for its high background autofluorescence and, indeed, using fluorescent secondary antibodies we could hardly detect peroxisomes. By using the recently introduced fluorochrome-labeled tyramines as substrates for HRP (
Peroxisomes in Fetal and Newborn Mouse Tissues
There have been only a few reports on peroxisomal proteins and their mRNAs in fetal and newborn mouse tissues. Because this report deals only with our findings in three major organs (liver, intestine, and skin), we discuss here our observations in the context of available literature. It is noteworthy that the overall distribution of mRNAs for CAT and AOX corresponds closely to the pattern reported for peroxisome proliferator-activated receptor- (PPAR
) (
regulates the transcription of peroxisomal ß-oxidation enzymes, particularly that of AOX 1 (
Liver
Peroxisomes in fetal rat liver were first described by routine electron microscopy (
Intestine
In the developing intestine of mouse fetus, the number of peroxisomes appears to increase with time, which is consistent with earlier cytochemical and biochemical reports (Pipan and Peni
nik 1975;
Skin
This is, to the best of our knowledge, the first report of epidermal localization of peroxisomal proteins by IHC and corresponding mRNAs by ISH. In agreement with biochemical data of CAT activity ( and other activators of nuclear hormone receptors (
In summary, the spatiotemporal distribution of peroxisomal proteins and corresponding mRNAs has been described during the fetal development of mouse liver, intestine, and skin. This was made possible by developing advanced protocols for nonradioactive ISH and IHC on paraformaldehyde-fixed and paraffin-embedded tissues, using improved enzyme substrates, antigen retrieval, and CARD to enhance detection sensitivity and subcellular microscopic resolution. Further studies using these techniques should be helpful in elucidation of cell-specific distribution of peroxisomaland also non-peroxisomalproteins and corresponding mRNAs during mouse development, particularly with regard to the wide field of transgenic and knockout mouse models.
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Acknowledgments |
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Supported by The Deutsche Forschungsgemeinschaft (grants Ba 1155/13).
We thank Drs U. Deschl and J. Pill, and G. Dietmann (Boehringer Mannheim; now Roche, Mannheim) for logistic support and for supplying pregnant mice, and Prof A. Völkl and Dr A. Schad for many helpful suggestions and discussions. The expert technical assistance of Heike Steininger and Richard Morlang is gratefully acknowledged.
Received for publication September 29, 2000; accepted October 4, 2000.
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