TECHNICAL NOTE |
Correspondence to: Akemi IshidaYamamoto, Dept. of Dermatology, Asahikawa Medical College, Asahikawa 078-8510, Japan.
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Summary |
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We describe an in situ DNA nick end-labeling method that can be performed at the electron microscopic level and can also be combined with immunoelectron microscopy. As the materials, we used skin tissues from normal skin and from Bowen's disease that had been cryofixed, freeze-substituted, and embedded in Lowicryl K11M resin. Ultrathin sections were cut and incubated with a reaction buffer containing digoxigenindUTP and terminal deoxynucleotidyl transferase. Digoxigenin nucleotides were labeled with anti-digoxigenin antibodies conjugated with colloidal gold. Specific signals were detected in the condensed chromatin of differentiated epidermal cells and hair follicles in normal skin and of dyskeratotic cells in Bowen's disease. The labeling density over chromosomal areas of apoptotic cells was significantly higher than that over chromosomal areas of mitotic cells or cytoplasmic areas. Ultrastructure was well preserved and double staining with an anti-keratin antibody was also successfully performed. This simple method has a wide range of applications to identify the nature of apoptotic cells and explore the mechanisms of apoptosis. (J Histochem Cytochem 47:711717, 1999)
Key Words: apoptosis, hair follicle, keratinocytes, skin, TUNEL
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Introduction |
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Electron microscopy is one of the most reliable methods to identify apoptotic cells in tissue sections (
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Materials and Methods |
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Normal human skin was obtained from the thigh and forehead of adults at the time of plastic surgery, and from the scalp of an aborted fetus at 21 weeks of gestational age. Tissue samples were also obtained from Bowen's disease during surgical operation. The skin tissues were cryofixed, cryosubstituted, and embedded in Lowicryl K11M resin (Chemische Werke Lowi; Waldkraiburg, Germany) according to methods previously described (
First we tested whether the Lowicryl K11M resin-embedded samples work for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL). TUNEL was performed at the light microscopic level using an Apop Tag peroxidase in situ apoptosis detection kit (S7100; Oncor, Gaithersburg, MD) according to the manufacturer's instructions, with some modifications. One-µm-thick Lowicryl resin-embedded sections were cut and mounted on silanized slides (Dako Japan; Kyoto, Japan). Some sections were fixed in 10% formalin in PBS, pH 7.4, for 10 min at room temperature (RT) and washed twice in PBS for 5 min each according to the manufacturer's recommendation. They were postfixed in ethanol:acetic acid 2:1 for 5 min at -20C and washed in two changes of PBS for 5 min each. Because we found that these fixation steps can be omitted without affecting the results, we did not fix other sections before the following procedures. Endogenous peroxidase was blocked by incubating in 2.0% hydrogen peroxide in PBS for 5 min. The sections were washed twice with PBS for 5 min each. Equilibration buffer was applied on the specimens for 1015 sec. The sections were incubated with a TdT enzyme mixture in reaction buffer at 37C for 1 hr. Specimens were rinsed in stop/wash buffer for 30 min at 37C and then in three changes of PBS for 5 min each. The sections were further incubated with anti-digoxigeninperoxidase for 30 min at RT. After washing twice in PBS for 5 min each, the sections were reacted with a mixture of 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Sigma; St Louis, MO) and 0.05% hydrogen peroxide for 2.5 min at RT. After washing in PBS, the sections were counterstained with Kernechtrot (Muto Pure Chemical; Tokyo, Japan) for 5 min and washed in PBS. This counterstain does not stain nuclei too strongly, so that nuclear signals of TUNEL became more evident. The sections were mounted in Geltol Mounting Medium (Lipshaw Immunon; Pittsburgh, PA). For negative controls, distilled water instead of TdT was mixed into the reaction buffer.
Next we tested whether colloidal gold can be used to detect digoxigeninnucleotides. Lowicryl K11M sections that were not fixed with formalin and ethanol:acetic acid were incubated with equilibration buffer as described above and then incubated with a TdT enzyme mixture in reaction buffer for 1060 min at 37C. Specimens were rinsed in stop/wash buffer for 30 min at 37C and then in three changes of PBS for 5 min each. Digoxigeninnucleotides were then labeled with an anti-digoxigenin sheep antibody conjugated with 10-nm gold (British BioCell; Cardiff, UK), which was diluted 10 times with Tris-buffered saline, pH 8.2, containing 1% bovine serum albumin, 1% normal goat serum, and 0.1% gelatin (Tris-wash/reaction buffer) at 37C for 30 min. The sections were washed twice with PBS and twice with distilled water for 5 min each. Subsequently, the gold particles were enhanced using an IntenSE silver staining kit (Amersham; Poole, UK). The sections were washed twice in distilled water for 5 min each, counterstained, and mounted as described above.
For in situ electron microscopic detection of fragmented DNA, ultrathin sections of Lowicryl-embedded tissues were cut and collected on formvar-coated nickel grids. After application of equilibration buffer for 1015 sec, the sections were incubated with a TdT enzyme mixture in reaction buffer at 37C for 1060 min. The specimens were rinsed in stop/wash buffer for 30 min at 37C. They were further washed twice with PBS and twice with Tris-wash/reaction buffer for 5 min each at RT. The sections were then incubated with anti-digoxigenin sheep antibody conjugated with 10-nm gold diluted 10 times with Tris-wash/reaction buffer at 37C for 30 min. The sections were washed twice with Tris-wash/reaction buffer and twice with distilled water for 5 min each at RT. They were stained with 1.5% uranyl acetate in methanol for 3 min at RT and observed in a transmission electron microscope.
For quantitative evaluations, numbers of gold particles per µm2 were counted over chromosomal areas of apoptotic cells, chromosomal areas of mitotic cells, and cytoplasm. As the material, Bowen's disease was used, because both mitosis and apoptosis were frequently observed in a single section in this disease and comparison of the labeling densities under the same experimental conditions was possible. For each compartment, 20 areas were randomly chosen on the photographs of electron microscopic in situ nick end-labeling.
For combination with immunoelectron microscopy, a monoclonal antibody to keratin K1 (34ßB4; Enzo Diagnostics, New York, NY) was used as a primary antibody. TUNEL was done as described above and the sections were further incubated with the K1 antibody diluted 20 times in PBS containing 1% bovine serum albumin, 1% normal goat serum, and 0.1% gelatin for 1 hr at 37C. The sections were washed twice in the same buffer and twice in Tris-wash/reaction buffer for 5 min each, followed by incubation with 15-nm gold-conjugated goat anti-mouse antibodies (BioCell) diluted 10 times in Tris-staining/wash buffer for 1 hr. Finally, the sections were washed twice with Tris-wash/reaction buffer and twice with distilled water for 5 min each. The sections were stained with uranyl acetate as described above. For immunohistochemistry, negative controls included incubation in the presence of the secondary antibody alone and incubation with unrelated primary antibodies.
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Results |
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At the light microscopic level, TUNEL on Lowicryl K11M-embedded normal human skin was successful by using both anti-digoxigeninperoxidase and colloidal gold-conjugated anti-digoxigenin (Figure 1). Positive reaction was observed in the differentiated epidermal keratinocytes (Figure 1A and Figure 1C) and inner hair root sheath cells (Figure 1D), as has been described previously (
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We then tried TUNEL at an ultrastructural level using colloidal gold-conjugated anti-digoxigenin as label, with successful results (Figure 2). The best results were obtained when the sections were incubated with TdT for 20 min as in the light microscopic study. The strong reactions were on the condensed chromatin of the differentiated epidermal keratinocytes and inner hair root sheath cells. Ultrastructure was satisfactory to identify subcellular structures, including nuclear membrane, mitochondria, keratin filaments, keratohyalin granules, and desmosomes.
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To clarify the difference between DNA ends generated by sectioning of the tissue blocks and apoptotic DNA breaks, we compared the labeling over the condensed chromatin masses in apoptotic (dyskeratotic) cells with that obtained in mitotic cells on sections of Bowen's disease. As shown in Table 1, the labeling density over chromosomal areas of mitotic cells was low, although it was still higher than that of cytoplasm, reflecting the DNA ends generated by sectioning. The labeling density over chromosomal areas of apoptotic or dyskeratotic cells was significantly higher than that of mitotic cells, demonstrating that the majority of the gold particles labeled DNA breaks due to apoptosis. Representative photographs of apoptotic and mitotic chromosomes in Bowen's disease are shown in Figure 3.
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We next tested whether the electron microscopic TUNEL could be combined with immunoelectron microscopy. A monoclonal antibody against K1 gave good immunoreactivity on the specimens stained for TUNEL (Figure 4).
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Discussion |
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Apoptosis is characterized by accompanying morphological changes, such as cell shrinkage, condensation of nuclear chromatin, and loss of microvilli (
Using this technique, we could correlate fine morphological changes with DNA fragmentation in apoptosis. Confident results were also obtained from materials that had been embedded in Lowicryl resin and kept at RT for more than 4 years. The preparation procedures for Lowicryl resin-embedded tissue blocks were the same as those that we and others have been using for immunoelectron microscopy over the past 10 years and have been proved to be suitable for detection of a number of molecular epitopes (
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to AI-Y (10470184), HT (08770618), and HI (08457233), and by a Grant from the Ministry of Health and Welfare, Japan to HI and a grant from Shiseido Co. to AI-Y.
We thank Dr Mori and Dr Yamada for providing us with fetal skin samples.
Received for publication August 20, 1998; accepted December 1, 1998.
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Literature Cited |
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