Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections

ELSEVIER

Journal of Virological Methods Journal of Virological Methods 52 (1995) 309-316

Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections Yoshito Eizuru a7*, Yoichi Minamishima a, Tadashi Matsumoto b, Toshinari Hamakado b, Mikio Mizukoshi b, Kazuyuki Nabeshima ‘, Masashi Koono ‘, Aichi Yoshida d, Hiroki Yoshida d, Masahiro Kikuchi e a Department of Microbiology, Miyazaki Medical College, Kihara, Kiyotake, Miyazaki 889-16, Japan ’ Ube Research Laboratory, Fujirebio Inc., Ushiake, Yoshiwa, Ube 759-01, Japan ’ Second Department of Pathology, Miyazaki Medical College, Kihara, Kiyotake Miyazaki 889-16, Japan ’ First Department of Pathology, Faculty of Medicine, Kagoshima University, 8-35-l Sakuragaoka, Kagoshima 890, Japan ’ First Department of Pathology, School of Medicine, Fukuoka Unhersity, Fukuoka, Japan

Accepted 20 October 1994

Abstract Non-isotopic in situ hybridization with a novel phenytoin (PHEl-labeled probe was developed. The mixture of cloned cytomegalovirus (CMV) DNA fragments was labeled by random primer technique using PHE-ll(spacer)-dUTP, instead of dTTP. The tissue sections were treated with 0.2 N HCl and with proteinase K (1 pg/ml), and then heated at 70°C in the presence of 50 or 75% formamide. The sections were hybridized with PHE-labeled probe at 37°C overnight. The hybridization signal was visualized by alkaline phosphatase-S-bromo-4-chloro-3-indolyl phosphate (BCIP)/4nitroblue tetazolium (NBT) system. Strong hybridization signals were detected in sections of the small intestine and the placenta, even when denatured in the presence of 50% formamide. In the case of small intestine, CMV DNA was also detected in the endothelial cells of the mucosa where apparent infected cell was not observed histologically. In the sections of the submaxillary gland, the lung, the adrenal gland and the ovary, hybridization signal was not

* Corresponding author. Present address: Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, 8-35-l Sakuragaoka, Kagoshima 890, Japan. Fax: + 81 (992) 75 5937. 0166-0934/95/$09.50

0 1995 Elsevier Science B.V. All rights reserved

SSDI 0166.0934(94)00163-4

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of Virological Methods 52 (1995) 309-316

detected when denatured in the presence of 50% formamide, but detected after denaturation with 75% formamide. Thus, in situ hybridization with the novel PHE-labeled probe is applicable to conventional formalin-fixed, paraffin-embedded tissue sections. Keyvords: Phenytoin; In situ hybridization; Cytomegalovirus

1. Introduction In situ hybridization depends on base-pairing between probe nucleic acids and target nucleic acids in cells or tissues. Originally, the probe was labeled with radioisotope (“H, “P, 35S or lz51) and the hybridization signal was visualized by autoradiography. In 1983, Brigati et al. introduced an in situ hybridization using biotin-labeled probe. Thereafter, a variety of non-radioactive labels (most of them are haptens) were employed, such as sulfone (Pezzella et al., 1987) dinitrophenyl (DNP) (Buchbinder et al., 1988), acetylaminofluorene (AFF) (Landegent et al., 1984) mercury-sulfhydryl-hapten (Hopman et al., 1987) digoxigenin (Yamada et al., 1989) bromodeoxyuridine (Niedobitek et al., 1988) and thymine dimer (Yamada et al., 1992). These non-radioactive probes have various advantages for routine applications: better resolution, shorter processing time, much easier detection of the hybridization signal, much longer probe half-life and safety. Phenytoin (PHE) is an anti-convulsive drug for epilepsy. Using PHE as a label, a novel non-radioisotope (non-RI) in situ hybridization technique was developed. This novel label system was examined for suitability for conventional formalin-fixed, paraffin-embedded tissue sections.

2. Materials

and methods

2.1. Preparation

of PHE-labeled

probe

The equimolar mixture of Eco RI D, E and G DNA fragments of human cytomegalovirus (HCMV), AD 169 strain, which had been cloned into lamda EMBLE4 was labeled with PHE by random primer labeling kit (Takara Shuzo Co., Kyoto) using PHE-ll(spacer)-dUTP, instead of dTTP. 2.2. Specimens

and pretreatment

of specimens

Specimens employed in this experiment were conventional formalin-fixed, paraffinembedded tissues. The tissue sections (5 pm thick) were fixed on organosilanated glass slides (Bokusui Brown Co., Tokyo). After deparaffination by treating with xylene, the sections were hydrated through alcohol series and dipped in phosphate-buffered saline (PBS, pH 7.4) for 5 min. The sections were sequentially treated with 0.2 N HCl at room temperature for 10 min and with proteinase K (1 pg/ml) in the presence of 2 mM CaCl, at 37°C for 15 min and then immersed in 50 or 75% formamide in 2 X SSC

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(1 X SSC = 0.15 M sodium chloride + 0.015 M sodium citrate) at room temperature for 10 min and denatured at 70°C for 15 min. Immediately after denaturation, the slides were dipped in ice-cold 50% formamide in 2 X SSC until hybridization. 2.3. Preparation

of cell pack

The HCMV-infected human embryonic lung fibroblasts (HEL) and non-infected HEL were collected by centrifugation at 1500 rpm for 5 min, and then fixed in 2% paraformaldehyde in PBS for 3 days and embedded in paraffin. After sectioning, the sections were pretreated as described above. 2.4. In situ hybridization The pretreated sections were solution consisting of 20 ng/ ml 10% dextran sulfate, 2 X SSC hybridization, the sections were

hybridized at 37°C overnight with the hybridization of heat-denatured PHE-labeled probe, 50% formamide, and 400 ng/ml of salmon sperm DNA. During the covered with cover glass and kept in a moist chamber.

2.5. Signal detection After hybridization, the sections were washed in 2 X SSC-0.1% SDS and in 0.5 X SSC-0.1% SDS for 5 min, respectively, and then dipped in 0.1 M Tris-HCl (pH 7.5)-0.15 M NaCl (buffer 1) for 2 min and incubated with 1% casein in buffer 1 for 10 min. The sections were reacted with alkaline phosphatase-labeled anti-PHE mouse monoclonal antibody (diluted to 1 : 25,000 in buffer 1) for 30 min. After washing twice in buffer 1 for 5 min each, the sections were dipped twice for 5 min each in 0.1 M Tris-HCl (pH 9.5)-50 mM MgCl,-0.1 M NaCl (substrate buffer). The sections were then reacted in a dark room for 24 h with 0.04% 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/O.O4% 4-nitroblue tetazolium (NBT) in substrate buffer. The reaction was stopped by dipping the sections in 10 mM Tris-HCl (pH 7.6)-l mM EDTA (TE buffer) for 5 min and mounted after washing with distilled water, with glycerol gelatin (Sigma Diagnostics, St. Louis, MO).

3. Results 3.1. In situ hybridization

in a model system

First, using paraformaldehyde-fixed, paraffin-embedded infected cell pack, we examined whether in situ hybridization with PHE-labeled probe was applicable to paraffinembedded tissue sections. The HCMV-infected HEL were collected by centrifugation and fixed in 2% paraformaldehyde in PBS for 3 days. After embedding in paraffin, the sections were hybridized with PHE-labeled probe. If the sections were not treated with 0.2 N HCl and/ or proteinase K, the hybridization signal was almost negligible (data not shown). However, when the sections were pretreated completely as described in Material and methods, a strong dark purple signal was detected in HCMV-infected cell

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Fig. 1. Hematoxylin/eosin

of Vimlogical Methods 52 (1995) 309-316

staining (a) and in situ hybridization

(b) in HCMV-infected

cell pack. X 200.

sections (Fig. lb), but no background was detected in non-infected cell sections (data not shown). This suggested that in situ hybridization with PHE-labeled probe is applicable to conventional pathological tissue sections. 3.2. In situ hybridization

in conventional

pathological

tissue sections of various organs

In situ hybridization with PHE-labeled probe was applied to conventional pathological sections of the small intestine, the placenta, the submaxillary gland, the lung, the adrenal gland and the ovary in which HCMV infection was histologically suspected.

Fig. 2. Hematoxylin/eosin

staining (a) and in situ hybridization

(b) in perforated

small intestine.

X 1.50.

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Fig. 3. Hematoxylin/eosin Fig. 2. X 100.

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of Virological Methods 52 (1995) 309-316

staining (a) and in situ hybridization

(b) in the mucosa of the same specimen

in

In the area of ulceration and penetration in the small intestine, many intranuclear and intracytoplasmic inclusions, typical of HCMV infection, were found in the endothelial cells throughout the submucosa, the muscularis propria and the subserosa (Fig. 2a). In these regions, a strong hybridization signal was detected (Fig. 2b). In addition, some endothelial cells in the mucosa were also positive for HCMV infection (Fig. 3b), while typical HCMV infection was not observed histologically (Fig. 3a). Thus, the expansion

Fig. 4. Hemotoxylin/eosin congenital HCMV infection.

staining X 200.

(a) and in situ hybridization

(b) in the placenta

from a baby with

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Fig. 5. Hemotoxylin/eosin

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staining (a) and in situ hybridization

(b) in the lung. x 200.

of the HCMV-infected area was recognized by in situ hybridization in comparison with histological examination. The placenta was obtained from a baby with apparent congenital HCMV infection. Histologically, the HCMV-infected cells were scattered in the section (Fig. 4a). In accordance with those infected cells, strong hybridization signals were detected (Fig. 4b). In spite of the presence of cytomegalic cells, in situ hybridization after denaturation

Fig. 6. Hemotoxylin/eosin

staining (a) and in situ hybridization

(b) in the adrenal gland. X 200.

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with 50% formamide did not give a positive signal in the sections of the submaxillary gland, the lung, the adrenal gland and the ovary. After denaturation in the presence of 75% formamide, a discernible, although not strong, hybridization signal was detected in the sections of these organs (Figs. 5 and 6).

4. Discussion The methodology of non-radioactive in situ hybridization has advanced considerably in recent years and improved as a result of the efforts of many investigators. The biotin-labeled probe made the detection of viral nucleic acids by in situ hybridization possible even for routine histological studies, although it was not as sensitive as radioactive in situ hybridization. With the use of the biotin-labeled probe, however, considerable problems may occur because of the presence of endogenous avidin-binding activity in frozen sections of several tissues, such as the liver, the kidney and the lymphoid organs (Banarjee and Pettit, 1984; Wood et al., 1981). Thus, the digoxigeninlabeled probe is gradually replacing biotin-labeling, because digoxigenin does not exist naturally in animals including human. We developed an in situ hybridization technique using a novel PHE-labeled probe which readily detected the viral nucleic acid in tissue culture. The PHE-labeled probe can be successfully applied to conventional formalin-fixed, paraffin-embedded tissue sections. However, the strength of the detection signal varied with specimens, which may be due to the degree of formalin-fixation. Too much fixation produced overcrosslinking of the double strand of viral DNAs and requires more energy to denature them. Indeed, a reasonable signal was obtained in some tissues when denatured in the presence of 75%, instead of 50%, formamide. If the denaturation was carried out at 100-105°C using a microwave oven as described by Unger et al. (1986) and Coates et al. (1987), a stronger signal may be obtained. Although the sensitivity of PHE-labeled probe was not compared with that of other non-RI-labeled probe, it was applicable to conventional formalin-fixed, paraffin-embedded tissue sections.

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