Journal of Histochemistry and Cytochemistry, Vol. 47, 711-717, May 1999, Copyright © 1999, The Histochemical Society, Inc.


TECHNICAL NOTE

Electron Microscopic In Situ DNA Nick End-labeling in Combination with Immunoelectron Microscopy

Akemi Ishida–Yamamotoa, Toshihiro Yamauchia, Hikaru Tanakaa, Hiroshi Nakanea, Hidetoshi Takahashia, and Hajime Iizukaa
a Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan

Correspondence to: Akemi Ishida–Yamamoto, Dept. of Dermatology, Asahikawa Medical College, Asahikawa 078-8510, Japan.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 digoxigenin–dUTP 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 47:711–717, 1999)

Key Words: apoptosis, hair follicle, keratinocytes, skin, TUNEL


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Electron microscopy is one of the most reliable methods to identify apoptotic cells in tissue sections (Wyllie et al. 1980 ). A multistage process of double-stranded DNA fragmentation is typically associated with morphological apoptosis. The in situ DNA nick end-labeling technique allows detection of fragmented DNA at the single-cell level (Gavrieli et al. 1992 ). Correlating ultrastructural changes with the DNA cleavage, Migheli et al. 1995 succeeded in detecting DNA breaks in apoptotic cells at the electron microscopic level. They obtained good ultrastructural preservation and specific labels with Araldite resin-embedded tissue samples. To further characterize the nature of apoptotic cells, combination with immunoelectron microscopy would be desirable. We have tested Lowicryl K11M resin for double staining of DNA cleavage and proteins and obtained satisfactory ultrastructural preservation to identify the cell types and strong signals for both DNA cleavage and immunohistochemistry. Here we present this technique with its potentially wide range of applications.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Shimizu et al. 1989 ; Ishida-Yamamoto et al. 1996 ).

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 10–15 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-digoxigenin–peroxidase 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 digoxigenin–nucleotides. 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 10–60 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. Digoxigenin–nucleotides 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 10–15 sec, the sections were incubated with a TdT enzyme mixture in reaction buffer at 37C for 10–60 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.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

At the light microscopic level, TUNEL on Lowicryl K11M-embedded normal human skin was successful by using both anti-digoxigenin–peroxidase 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 (Gavrieli et al. 1992 ; Tamada et al. 1994 ). Because we obtained relatively high background in colloidal gold labeling, we shortened the incubation time with TdT enzyme and found that 20 min of incubation gave the best positive reactions with minimal background signals on the cytoplasm. There was no positive reaction in negative controls (Figure 1B).



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Figure 1. TUNEL on Lowicryl K11M-embedded samples. Stained with anti-digoxigenin–peroxidase visualized with diaminobenzidine (A,B) or with a silver-enhanced 10-nm gold-conjugated anti-digoxigenin antibody (C,D). There are positive cells (arrowheads) just beneath the epidermal cornified layer in A and C but not in B (a negative control). Positive cells are also seen in the inner hair root sheath of fetal scalp skin (D). The dark pigment in the epidermal basal layer, around hair papilla (p), and in the hair shaft is melanin. Bar = 10 µm.

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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Figure 2. Electron microscopic TUNEL using Lowicryl resin. A differentiated epidermal keratinocyte (A) and inner hair root sheath cells (B,C). The marked square areas in A and B are shown in higher magnifications in the inset to A and in C, respectively. Signals are seen on the condensed nuclear chromatin (*). Degenerative nuclear membrane is marked with an arrowhead. Bars: A,C = 0.1 µm; B = 0.5 µm.

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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Figure 3. Comparison between TUNEL over condensed chromatin (*) in apoptotic nucleus and mitotic nucleus; section of Bowen's disease. The marked square areas in A and C are shown at higher magnification in B and D, respectively. There is some label on the mitotic nuclei (D), but much more labeling is seen on the apoptotic nuclear chromatin (B). Bars: A,C = 1 µm; B,D = 0.1 µm.


 
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Table 1. Gold particles (number of particles/µm2) over different structural compartments in Bowen's diseasea

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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Figure 4. A combination of TUNEL and immunoelectron microscopy. Differentiated epidermal cells. TUNEL signal (10-nm gold) is observed at the condensed nuclear chromatin (*) surrounded by degenerative nuclear membrane (arrowhead). K1 staining (15-nm gold) is seen on keratin filaments in the peripheral area of the apoptotic cell cytoplasm (arrow) and in a cornified cell (C). Bar = 0.1 µm.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Apoptosis is characterized by accompanying morphological changes, such as cell shrinkage, condensation of nuclear chromatin, and loss of microvilli (Wyllie et al. 1980 ). The biological hallmark of apoptosis is the cleavage of chromosomal DNA that can be detected by TUNEL (Figure 1). We have presented methods that enable us to perform TUNEL at the electron microscopic level (Figure 2 and Figure 3) and that can be combined with immunoelectron microscopy (Figure 4). This method also detected DNA ends probably generated by sectioning (Table 1; Figure 3), and this is in agreement with previous studies using a similar in situ labeling technique (Thiry 1992 ). However, the labeling density over condensed chromosomes of apoptotic cells was much higher than that generated by sectioning typically seen over mitotic chromosomes (Table 1; Figure 3), and therefore identification of apoptotic nuclei was not hampered.

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 (Shimizu et al. 1989 , Shimizu et al. 1992 ; Ishida-Yamamoto et al. 1991 , Ishida-Yamamoto et al. 1996 , Ishida-Yamamoto et al. 1997 ). By combining TUNEL and immunohistochemistry at the electron microscopic level, the series of events involved in apoptosis were effectively revealed, thereby leading to better understanding of the mechanisms of apoptosis.


  Acknowledgments

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.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501[Abstract]

Ishida–Yamamoto A, Eady RAJ, Watt FM, Roop DR, Hohl D, Iizuka H (1996) Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis. J Histochem Cytochem 44:167-175[Abstract/Free Full Text]

Ishida–Yamamoto A, McGrath JA, Chapman SJ, Leigh IM, Lane EB, Eady RAJ (1991) Epidermolysis bullosa simplex (Dowling-Meara) is a genetic disease characterized by an abnormal keratin filament network involving keratins K5 and K14. J Invest Dermatol 97:959-968[Abstract]

Ishida–Yamamoto A, McGrath JA, Lam H, Iizuka H, Friedman RA, Christiano AM (1997) The molecular pathology of progressive symmetric erythrokeratoderma: a frameshift mutation in the loricrin gene and perturbations in the cornified cell envelope. Am J Hum Genet 61:581-589[Medline]

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Shimizu H, Ishida–Yamamoto A, Eady RAJ (1992) The use of silver-enhanced 1-nm gold probes for light and electron microscopic localization of intra- and extracellular antigens in skin. J Histochem Cytochem 40:883-888[Abstract/Free Full Text]

Shimizu H, McDonald JN, Kennedy AR, Eady RAJ (1989) Demonstration of intra- and extracellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immunoelectron microscopy. Arch Dermatol Res 281:443-448[Medline]

Tamada Y, Takama H, Kitamura T, Yokochi K, Nitta Y, Ikeya T, Matsumoto Y (1994) Identification of programmed cell death in normal human skin tissues by using specific labelling of fragmented DNA. Br J Dermatol 131:521-524[Medline]

Thiry M (1992) Highly sensitive immunodetection of DNA on sections with exogeneous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues. J Histochem Cytochem 40:411-419[Abstract/Free Full Text]

Wyllie AH, Kerr JFR, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251-306[Medline]