Immunohistochemical Detection of Human Gastrointestinal Glutathione Peroxidase in Normal Tissues and Cultured Cells with Novel Mouse Monoclonal Antibodies

Abstract

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.

Keywords

gastrointestinal glutathione peroxidase monoclonal antibody immunohistochemical staining Western blotting gastric carcinoma cell

The glutathione peroxidase family (GPx) are selenoenzymes that catalyze the reduction of hydrogen peroxide, organic hydrogen peroxides, or lipid peroxides in the presence of glutathione and function to detoxify peroxides in the cell (Thomas et al. 1990; Esworthy et al. 1991; Ursini et al. 1995). This activity depends on the expression of multiple isozymes. Three of four isozymes, the classical cellular GPx (cGPx, GPx1), the extracellular GPx (eGPx, GPx3), and the phospholipid hydroperoxide GPx (phGPx, GPx4), were previously purified from tissues and characterized at the protein level (Rotruck et al. 1973; Maddipati et al. 1987; Takahashi et al. 1987; Maiorino et al. 1990). With the exception of phGPx, a monomer, all GPx enzymes are comprised of four identical subunits (monomer molecular weight 19–25 kD). Each subunit contains a molecule of selenocysteine (Cys^Se) localized within the enzyme active site. The Cys^Se is believed to participate directly in electron donation to the peroxide substrate and becomes oxidized in the process. The enzyme then uses glutathione as an electron donor to regenerate the reduced form of Cys^Se (Ursini et al. 1995). cGPx and phGPx are present in most human tissues examined to date. cGPx is abundantly present in erythrocytes, kidney, and liver (Frampton et al. 1987), and phGPx is present in testis at a high level (Roveri et al. 1992). In contrast, eGPx exhibits tissue-specific expression, and is detected in plasma, milk and lung (Takahashi et al. 1987; Maiorino et al. 1990; Avissar et al. 1991; Yoshimura et al. 1991).

In addition to these three isozymes, a novel selenium-dependent GPx was characterized by expressing a cDNA isolated from human hepatoma HepG2 cells. The mRNA was expressed in gastrointestinal tissues but not in other human tissues such as kidney, heart, lung, placenta, or uterus. Therefore, the new GPx was designated gastrointestinal GPx (giGPx, GPx2) (Akasaka et al. 1990; Chu et al. 1993). cGPx and giGPx display similar substrate specificities; both catalyze the reduction of hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide, but not of phosphatidylcholine hydroperoxide, in the presence of glutathione.

However, the localization of giGPx protein in human tissues and its biological characteristics, other than its enzymatic activity, remain unknown. As a first step to resolve these questions, we attempted to establish mouse hybridomas that produce a monoclonal antibody (MAb) against human giGPx. In this article, we describe the characterization of MAbs against human giGPx, from mice immunized with a giGPx-derived peptide. Moreover, we demonstrate human giGPx protein expression in stomach, small intestine, large intestine, liver and gallbladder by Western blotting and immunohistochemical staining techniques using the human giGPx MAbs.

Materials and Methods

Cells

KATO-III (Sekiguchi et al. 1978), HuG-1 (Imanishi et al. 1990), OCUM1 (Kubo 1991), and Okajima (Nakajima et al. 1987) are human gastric carcinoma cell lines and TAB089 (Yang et al. 1989) is a B-lymphoblastoid cell line. The KATO-III cell line was obtained from the Japanese Cancer Research Resources Bank. These cell lines were cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum and antibiotics at 37C in a humidified atmosphere of 5% CO₂.

giGPx mRNA expression in KATO-III, OCUM-1, and TAB089 cells was assayed by RT-PCR. giGPx mRNA expression was detected in KATO-III and OCUM-1 cells, but not in TAB089 cells (data not shown).

Synthesis of Human giGPx-derived Peptides

The human giGPx-derived peptide (SAISLDGEKVDFNTFRGRAC) (Akasaka et al. 1990) synthesized for this study was highlighted by the algorithm (Hopp and Woods 1981) which searches for a local maximum in a hydrophobicity profile smoothed over five residues. The peptide corresponds to the giGPx original sequence plus an additional C-terminal Cys residue to allow conjugation with carrier protein (see below). The peptide was synthesized by the solid-phase method (Merrifield 1969) using Fmoc chemistry (Fields and Noble 1990) on a peptide synthesizer (Model PSSM8; Shimazu Seisakujo, Kyoto, Japan). After cleavage from the resin, the peptide was lyophilized and its purity was tested by analytical HPLC using a Shim-pack PREP-ODS column (4.6 × 150 mm; Shimazu Seisakujo). The purity of peptide preparations exceeded 95%.

Immunization of Mice

The giGPx-derived peptide described above was conjugated with maleimide-activated keyhole limpet hemocyanine (Pierce; Rockford, IL). BALB/C mice were injected IP with 50 μg of the immunogen emulsified with Freund's complete adjuvant. Mice were subsequently injected IP on a further four occasions at 2-week intervals with 50 μg immunogen emulsified with Freund's incomplete adjuvant. After 2 more weeks, mice were injected IV with 50 μg of immunogen in PBS. Splenic lymphocytes were isolated from the mice 3 days after the final booster and fused with a SP2/0-Ag14 mouse myeloma cell line as previously described (Galfre et al. 1977).

Enzyme-linked Immunosorbent Assay

ELISA was based on a modification of a previously described protocol (Komatsu et al. 1990). The giGPx-derived peptides were used as antigen. Wells were coated at 4C overnight with antigen (50 ng/well) in 50 μl of 0.05 M carbonate buffer (pH 9.6). Each well was washed with washing buffer (TPBS) (0.05% v/v Tween-20, 0.01 M PBS, pH 7.4) and was incubated with PBS containing 20% (v/v) Block-Ace (Dainihon Seiyaku; Suita, Japan) at room temperature (RT) for 2 hr. After washing with TPBS, 50 μl of culture fluid was added to each well and the plate was incubated at RT for 1 hr. After washing with TPBS, each well was incubated with peroxidase (POD)-conjugated anti-mouse IgG (American Qualex; San Clemente, CA) (50 μl/well) at RT for 30 min. The wells were washed with TPBS and incubated with a substrate mixture (0.1% w/v o-phenylenediamine, 0.015% v/v hydrogen peroxide, 0.5 M citric acid-disodium phosphate buffer (pH 5.0) (100 μl/well) at RT for 30 min. The reactions were stopped by the addition of 2 N H₂SO₄ (50 μl/well) and color yield was monitored at 490 nm (Model 550; Nippon Bio-Rad Laboratories; Tokyo, Japan).

Western Blotting Assay

Cell extracts were obtained from 1 × 10⁸ cultured cells in 1 ml of low-salt lysis buffer [10 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 3 mM MgCl₂, 1 mM dithiothreitol, 1 mM phenylmethyl sulfonyl fluoride, and 0.5% (v/v) NP-40] on ice for 30 min. For preparation of human tissue extracts, tissue samples were homogenized and treated with low-salt lysis buffer on ice for 30 min. After centrifugation at 10,000 × g for 10 min at 4C to remove cell debris, lysates were fractionated by SDS-PAGE on a 12.5% (w/v) polyacrylamide gel (Laemmli 1970) and fractionated proteins transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (ATTO; Tokyo, Japan) using the methods of Towbin et al. (1979) and Burnette (1981). Loading of cell extract for SDS-PAGE was corrected for levels of β-actin (by Western blotting) using 5 × 10⁵ KATO-III cells as the 100% standard. The PVDF membrane was treated with Block-Ace at 4C overnight and then with mouse MAb at RT for 45 min. After washing with TPBS, the membrane was treated with POD-conjugated anti-mouse IgG, after which the membrane was washed and the MAb reaction visualized using the ELC Western blotting detection kit (Amersham Pharmacia Biotech; Tokyo, Japan).

Figure 1

Reactivity of anti-human giGPx MAbs in Western blotting assays. (A) KATO-III (K) and TAB089 (T) cell lysates were separated electrophoretically and blotted onto PVDF membrane as described in Materials and Methods. The membrane was incubated with anti-human giGPx MAbs (FEG-1, -2, -3, and -4) and anti-HIV transmembrane protein MAb (anti-HIV MAb) (Komatsu et al. 1991). As a negative control for nonspecific binding of secondary antibody (–), KATO-III cell lysate was treated with POD-conjugated anti-mouse IgG antibody without primary antibody. Cell lysate loading for SDS-PAGE was adjusted to the concentration of β-actin in KATO-III cells (5 × 10⁵ cells/lane). (B) KATO-III cell lysates (5 × 10⁵ cells/lane) were separated electrophoretically and blotted onto PVDF membrane as described in Materials and Methods. The membrane was incubated with FEG-1 MAb preabsorbed with the giGPx-derived synthetic peptide used for immunization of mice. Lane 1, no peptide; Lanes 2–5, peptide at 1 ng/ml, 100 ng/ml, 10 μg/ml, and 1 mg/ml, respectively.

Immunohistochemical Staining

Cultured cells were fixed with 10% (v/v) phosphate-buffered formalin (pH 7.2), and resuspended in warmed 2% (w/v) agar solution after centrifugation at 1000 rpm for 5 min. Cells were centrifuged at 3000 rpm for 5 min and cooled for 30 min at 4C. The cell pellets within solid agar were again fixed with phosphate-buffered formalin and embedded in paraffin. A variety of human tissues to be analyzed for giGPx localization were selected from surgically removed or autopsy materials (patient age 45–75 years), fixed with phosphate-buffered formalin, and embedded in paraffin. Immunohistochemical staining was performed using the standard labeled streptavidin–biotin–peroxidase complex method (LSAB kit; Dako Japan, Kyoto, Japan). Briefly, after routine deparaffinization, 4-μm-thick sections were pretreated with 0.3% (v/v) H₂O₂ in methanol for 30 min and incubated with anti-human giGPx MAb overnight in a moist chamber at 4C. For negative controls, normal mouse serum (diluted 1:100) was used instead of primary antibody. Counterstaining of nuclei was achieved with 0.3% (w/v) methyl green solution.

Results

Generation of Hybridomas and Specificity of MAbs

Immune splenocytes were fused with SP2/0-Ag14 murine myeloma cells using polyethylene glycol 2000, and hybridoma cells were generated in hypoxanthine/aminopterin/thymidine culture medium. Hybridoma supernatants were screened for anti-giGPx activity by ELISA, using a giGPx-derived synthetic peptide as antigen. Four anti-giGPx-positive hybridomas secreted antibodies which were specific for a 24-kD protein identified by Western blotting using KATO-III cell lysate as antigen. The apparent molecular weight of this protein corresponded to that predicted from the amino acid sequence of human giGPx. The hybridomas were subcloned twice by the limiting dilution method and all four hybridoma clones were found to secrete IgG1 antibodies.

The four MAbs, denoted FEG-1, -2, -3, and -4, were tested by Western blotting for reactivity with KATO-III and TAB089 cell lysates. All MAbs recognized a 24-kD protein in KATO-III cell lysate but not in TAB089 cell lysate (Figure 1A). Whereas FEG-1 and -4 MAbs reacted strongly with KATO-III cell lysate, FEG-2 and -3 MAbs showed weak reactivity. No reactivity was observed with anti-HIV transmembrane protein MAb (Komatsu et al. 1991) and POD-conjugated anti-mouse IgG as secondary antibody in KATO-III cell lysate.

As a control for antibody specificity, antibody was pretreated with the giGPx-derived synthetic peptide for 1 hr at 37C and the effect on reactivity with KATO-III lysate observed by Western blotting. A typical pattern using FEG-1 MAb is shown in Figure 1B. Reactivity with the 24-kD protein in KATO-III cell lysates was lost with increasing peptide concentration up to 10 μg/ml, strongly suggesting that the 24-kD protein in KATO-III cells has the same antigenicity as the peptide.

Figure 2

Crossreactivity of anti-human giGPx MAbs. (A) KATO-III (K), TAB089 (T) cell lysates, and commercial cGPx protein derived from human erythrocytes (cG: 5 μg protein/lane) were resolved by SDS-PAGE and blotted onto PVDF membrane as described in Materials and Methods. One of two cGPx protein lanes was separated after transfer to PVDF for protein visualization with Amido black 10B (Lane PS). The remaining PVDF membrane was incubated with anti-phGPx MAbs or FEG-1 MAb as indicated, then developed as described in Materials and Methods. Cell lysate loading for SDS-PAGE was adjusted to the concentration of β-actin in KATO-III cells (5 × 10⁵ cells/lane). (B) KATO-III cell lysates (5 × 10⁵ cells/lane) (K) and human plasma (5 μl/lane) (P) were separated electrophoretically and blotted onto PVDF membrane as described in Materials and Methods. The membrane was incubated with FEG-1 and anti-human eGPx MAb.

The crossreactivity of mAbs was investigated by Western blotting using a commercially available human cGPx protein (Sigma Chemical; St Louis, MO), an anti-phGPx MAb. The anti-phGPx MAb specifically recognized the 22-kD phGPx protein in lysates of KATO-III and TAB089 cells but did not recognize the 24-kD protein that was detected with the FEG series of MAbs (Figure 2A). Moreover, the FEG series of MAbs did not crossreact with large amounts of the commercial cGPx protein which although also a 24-kD protein, migrates slightly faster than putative human giGPx, as visualized by Amido Black 10B staining. For example, the reactivity of FEG-1 MAb with the commercial cGPx protein is shown in Figure 2A.

Next, the crossreactivity with eGPx was investigated by using a commercially available anti-eGPx mAb (Seikagaku Kogyo; Tokyo, Japan). Although the anti-eGPx MAb recognized a 23-kD protein in human plasma, it did not detect the 24-kD protein in KATO-III cell lysate (Figure 2B). Several bands at 26–120 kD were faintly detected in human plasma with the FEG series of MAbs and the anti-eGPx MAb, but similar bands were also detected in the absence of primary antibody (data not shown). These results strongly suggest that the four FEG MAbs specifically recognize human giGPx without crossreactivity to human phGPx, eGPx, or cGPx antigens. We therefore define FEG MAbs -1, -2, -3, and -4 as anti-human giGPx MAbs. FEG-1 mAb (reacting most strongly with giGPx antigen; see Figure 1A) was used for all subsequent experiments.

Reactivity with Human Tissues as Assayed by Western Blotting

The reactivity of FEG-1 MAb with giGPx antigen in normal human tissues was investigated by Western blotting using 29 human tissue extracts as antigen (Table 1). Although giGPx antigen was readily detected in stomach, duodenum, large intestine, liver, and gallbladder, it was not detected in other tissues such as salivary gland and esophagus. The reactivity of FEG-1 MAb with liver and gallbladder tissue was stronger than that with stomach, duodenum, and large intestine. Perhaps surprisingly, giGPx antigen was not detected in a tissue extract from pancreas. It is conceivable, however, that giGPx was degraded by pancreatic enzyme activity during preparation of the tissue extract, a hypothesis supported by the detection of little β-actin in extracts of fresh pancreas in a control Western blotting assay using an anti-β-actin antibody. The data strongly indicate that human giGPx is expressed specifically in the tissues of the gastrointestinal tract.

Immunohistochemical Localization of giGPx in Human Tissues

Immunohistochemical staining using FEG-1 MAb showed giGPx localization mainly to the neck area of the gastric pits and the basal portion of the crypts in duodenum, ileum, and large intestine (Table 1; Figure 3). giGPx was found in both cytoplasm and nucleus of epithelial cells, the latter showing rather stronger positivity than the former. The intensity was relatively weaker in the large intestine compared to the small intestine, particularly in the ileal mucosa. giGPx was observed in the epithelium of bile duct, pancreatic duct, and salivary gland tissue. Furthermore, positive staining was also seen in the lower portion of the mucosal folds, particularly at the Rokitansky–Aschoff sinus of the gallbladder. Liver cells showed a relatively weak reaction in the centrilobular area. FEG-2, -3, and -4, were also used for immunohistochemical studies on human tissues. Unlike FEG-1 MAb, some artifacts were detectable using the other FEG MAbs (data not shown). However, the pattern of reactivity with giGPx closely paralleled that observed for FEG-1 MAb. It may be that the specificity of the FEG-2, -3, and -4 MAbs is lower than that of FEG-1 MAb in formalin-fixed samples.

Table 1

Reactivity of FEG-1 MAb with human tissues by Western blotting and immunohistochemical staining a

	Reactivity
Tissue	Western blotting b	Immunohistochemical staining
Submandibular gland	–	1 + Duct
Esophagus	–	–
Stomach	1 + to 2 +	2 +
Duodenum	1 +	3 +
Ileum	ND	3 +
Large intestine	2 +	1 + to 2 +
Liver	3 +	1 + to 3+ Bile duct 3+; liver cells 1 +
Gallbladder	3 +	2 +
Pancreas	ND	1+ Small duct
Cerebrum	–	-
Cerebellum	–	-
Lung	–	-
Heart muscle	–	-
Aorta	–	-
Spleen	–	-
Bone marrow	–	-
Fatty tissue	–	-
Pituitary gland	ND	1+ Intermediate lobe, focal
Thyroid gland	–	–
Thymus	ND	1+ Hassal body, slightly positive
Adrenal cortex	–	–
Adrenal medulla	–	–
Kidney	–	–
Testis	–	–
Prostate gland	–	–
Ovary	ND	–
Endometrium	ND	–
Uterine cervix (ecto-, endo-)	ND	–
Breast	ND	–
KATO-III	3 +	3 +
TAB089	–	–

^aIntensity of positivity; -, not detectable; 1+, low; 2+ medium; 3+, high; ND, not determined.

^bTissue extract loading for SDS-PAGE was ajdusted for the concentration of β-actin in KATO-III cells (5 × 10⁵ cells/lane).

Reactivity with Other Gastric Carcinoma Cell Lines and Localization of giGPx in Cells

The reactivity of FEG-1 MAb with three gastric carcinoma cell lines, Okajima, OCUM1, and HuG-1 was investigated by Western blotting (Figure 4). giGPx was detected as a 24-kD protein in lysates of all three cell lines, as well as in KATO-III. This suggests that similar forms of giGPx are stably expressed in normal gastrointestinal tissue and in gastrointestinal carcinomas. To confirm the localization of giGPx in cells, each of the three gastric carcinoma cell lines was stained immunohistochemically using FEG-1 MAb. Both the nuclei and cytoplasm of Okajima cells were strongly stained (Figure 5A). Similar results were obtained with OCUM1, HuG-1, and KATO-III cells (data not shown). Twenty to 60% of total cells were immunohistochemically positive, depending on the cell line. When normal mouse serum (diluted 1:100) was used instead of FEG-1 MAb as a negative control, giGPx-positive cells were not detected (data not shown). Moreover, no expression of giGPx was observed in TAB089 cells, a B-lymphoblastoid cell line (data not shown).

The reactivity of FEG-1 preincubated with its peptide (10 μg/ml) for 1 hr at 37C was tested on Okajima cells (Figure 5B). In confirmation of the effect seen on Western blotting (Figure 1B), pretreatment of FEG-1 with the peptide used to raise it completely negated any interaction. Taken together, these data strongly suggest that FEG-1 MAb is indeed specific for human giGPx.

Discussion

We established four hybridoma clones producing MAbs against human giGPx antigen. These mAbs did not crossreact with commercial human cGPx, human ph-GPx, or human eGPx, suggesting that p24 detected in KATO-III cell lysate using the MAbs described herein is indeed likely to be giGPx. On the basis of the evidence presented here, we therefore propose that the four MAbs described specifically interact with human giGPx.

The amino acid sequence of human giGPx shares 95% homology with its mouse counterpart (Chu et al. 1996). The 19-amino-acid peptide used for mouse immunization in this study shows 89% homology with the corresponding mouse sequence. The high degree of homology between mouse and human giGPx would be predicted to cause difficulties with raising a titer of mouse antiserum against the human giGPx peptide. Indeed, p24 giGPx observed in KATO-III cell lysate was not detected when antisera from the immunized mice were used in a Western blotting assay (data not shown). Moreover, the titer of ascites-type MAbs was lower than one of hybridoma culture supernatant in a Western blotting assay (data not shown), suggesting possible neutralization with mouse intraperitoneal giGPx. Attempts to produce a monospecific rabbit antiserum against the same peptide readily resulted in high antibody titer for a period of 1 month (unpublished data), indicating that the human giGPx-derived synthetic peptide described here possesses an epitope(s) with relatively stronger antigenicity in rabbits than in mice. Chu et al. (1990) have also reported another monospecific rabbit serum against a synthetic peptide corresponding to C-terminal human giGPx. The report suggests that it is possible to establish hybridomas producing MAbs against the epitope(s) of the C-terminal human giGPx.

Figure 3

Immunohistochemical detection of giGPx in normal human tissues. Stomach, ileum, ascending colon, and liver tissue were stained with FEG-1 MAb as described in Materials and Methods. giGPx stained positive in the nuclei and cytoplasm of cells at the neck of gastric pits in pyloric gland mucosa (A), at the crypts of ileal (B) and colonic mucosa (C), and in bile duct epithelia in the liver (D). Hepatocytes showed low interaction in the centrilobular area (arrows) (E). In general, the intensity of staining is rather stronger in nucleus than in cytoplasm. Bars: A,C–E = 50 μm; B = 25 μm.

The human giGPx mRNA has been isolated from human liver, large intestine, and human hepatoma HepG2 cDNA libraries (Chu et al. 1993). We detected giGPx in stomach, small intestine, large intestine, liver, and gallbladder tissue, as evidenced by Western blotting. Esworthy et al. (1998) showed that giGPx protein was produced in the mucosal epithelium of the gastrointestinal tract in adult rats. These findings were confirmed by our immunohistochemical studies, which showed giGPx localization mainly in the cytoplasm and nuclei of mucosal epithelia at the basal area of the gastric pits and intestinal crypts. Taken together, this evidence strongly suggests that giGPx is specifically expressed in the gastrointestinal tract.

Figure 4

Reactivity of anti-human giGPx MAbs with gastric carcinoma cell lines in Western blotting assays. Cell lysates of three gastric carcinoma cell lines in addition to KATO-III and TAB089 were electrophoretically separated and blotted onto PVDF membrane as described in Materials and Methods. The membrane was then treated with FEG-1 MAb and developed as described in Materials and Methods. Cell lysate loading for SDS-PAGE was adjusted to the concentration of β-actin in KATO-III cells (5 × 10⁵ cells/lane).

Godeas et al. (1996) demonstrated the specific localization of phGPx in nuclear and mitochondrial fractions of rat testis. Imai et al. (1998) also described the subcellular localization of phGPx in phGPx-over-expressing cells. phGPx was predominantly found in nuclear fractions in these reports. However, Chu et al. (1993) showed giGPx to be present mainly in cytosolic fractions of giGPx-transfected cells. In the immunohistochemical work outlined here, giGPx was found to be expressed in both the cytoplasm and the nucleus of gastric carcinoma cells. On this basis, it is tempting to speculate that giGPx plays a part in the redox regulation of specific transcription factors after translocation into the nucleus in gastrointestinal tract cells. The novel MAbs described for the first time in this work show high specificity for giGPx in a range of human tissues, as evidenced by Western blotting and immunohistochemical staining techniques.

Figure 5

Immunohistochemical staining of Okajima cells. (A) The gastric carcinoma cell line Okajima was immunohistochemically stained with FEG-1 MAb as described in Materials and Methods. giGPx stained positive in both cytoplasm and nuclei of cancer cells. (B) As in A, except that FEG-1 MAb was preabsorbed with the giGPx-derived synthetic peptide used for immunization of mice. Counterstaining of nuclei was achieved with methyl green solution. Bars = 10 μm.

Footnotes

Acknowledgements

Supported in part by a grant from Kitasato University Graduate School of Medical Sciences (No. 9901).

We wish to thank Ms K. Hana and Ms Y. Numata for technical support and Dr M. Rau for critical reading of the manuscript.

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