Acta Histochem. Cytochem. 36 (2): 135–144, 2003

Immunohistochemical Distribution of Human Basigin by Using a Novel Monoclonal Antibody

Katsuya Hiraishi1, Tatsuo Ide1, Fumie Jimma1, Hiroshi Ohi1, Fumiko Inokuchi1, Teruo Miyauchi1 and Keiji Suzuki2

1Japan Immunoreseach Laboratories, Co., Ltd., 351–1 Nishiyokote-cho, Takasaki, Gunma 370–0021, Japan and 2Division of Histopathology, Department of Laboratory Sciences, Gunma University School of Health Sciences, 3–39–15 Showamachi, Maebashi, Gunma 371–8514, Japan

Received February 15, 2003; accepted March 6, 2003

Basigin is a member of the immuno- cells. Excellent immunostaining results globulin (Ig) superfamily which has were obtained with formalin-fixed tissues strong homology with both Ig V domain after the antigen retrieval procedure. and the -chain of the major histocom- Our immunohistochemical observations patibility complex (MHC) class II antigen. revealed that basigin is widely expressed Human basigin is also known as M6 in various organs, but on specific cell antigen, neurothelin and EMMPRIN types, such as blood capillaries of the (extracellular matrix metalloproteinase brain, proximal convoluted tubules of inducer). In this study, we developed a the kidney, cardiac muscle of the heart, novel anti-human basigin monoclonal trophoblasts of the placenta and basal antibody, J-BIT-1 and used it to map out cells of the stratified squamous epithe- the distribution of basigin in human lium. The location of basigin is the cell tissues. The specificity of J-BIT-1 was surface, especially the site of cell adhe- confirmed by western blot analysis and sion. These observations suggest that immunocytochemical techniques utiliz- basigin might be involved in basic cell ing basigin cDNA transfected L cells. J- functions which are promoted with cell- BIT-1 appropriated to immunohistochem- cell and cell-extracellular matrix inter- ical staining for formalin-fixed, paraffin- action. embedded tissues and methanol-fixed

Key words: basigin, monoclonal antibody, immunohistochemistry, cell adhesion

I. Introduction such as M6 antigen [14], human neurothelin (CD147 anti- gen) [17, 27] and EMMPRIN (extracellular matrix metallo- Basigin is a member of the immunoglobulin (Ig) super- proteinase inducer) [4]. Chicken basigin termed HT7 or family, which has two Ig like domains [20–22]. These Ig like neurothelin is expressed in the blood capillaries in the domains have strong homology with both Ig V domain and brain, and it is believed that CD147 antigen is involved in the -chain of the major histocompatibility complex (MHC) the formation of blood-brain barrier (BBB) [13, 27, 29]. Fur- class II antigen [20–22]. This transmembrane glycoprotein, ther, it is thought that human basigin, termed EMMPRIN is basigin, was found and cloned by cDNA library from F9 expressed in the cancer cell surface and is involved in the mouse embryonal carcinoma cells as the carrier protein of induction of matrix metalloproteinase from the fibroblast Lotus tetragonolobus agglutinin (LTA) receptor(s) [20]. [4, 15, 19]. Basigin homologous molecules have been independently It has been reported that a knock out mouse targeting reported under other names [21, 22, 30], such as chicken the basigin gene showed abnormalities of body size, HT7 [29], mouse gp42 [2], rat OX-47 [8], and chicken 5A11 memory function, female reproduction, spermatogenesis, [7]. Human basigin has also been known under other names, lung tissue structure, liver tissue structure, mixed lympho- cyte culture response to allogeneic spleen cells, retinal Correspondence to: Katsuya Hiraishi, Japan Immunoreseach Labora- function and photoreceptor cell maintenance [10–12, 18, 23]. tories, Co., Ltd. (JIMRO), 351–1 Nishiyokote-cho, Takasaki, Gunma However, at present, the exact roles of this multi-functional 370–0021, Japan. glycoprotein, basigin, is unclear. We thought that the distri-

135 136 Hiraishi et al. bution of basigin will provide clues to its functions. In the present study, we developed a novel anti-human basigin monoclonal antibody, named J-BIT-1 which can be applied to immunostaining for formalin-fixed, paraffin- embedded tissues. Immunohistochemical studies were per- formed to map out the distribution of basigin in human tissues by using this novel antibody, J-BIT-1.

II. Materials and Methods Preparation of fusion proteins A glutathione S-transferase (GST)-basigin fusion pro- tein and a series of maltose binding protein (MBP)-basigin fusion proteins were utilized in the present studies. By using GST Gene Fusion System (Pharmacia), the GST-basigin fusion protein containing human basigin (28 kDa) domain [21] and GST (25 kDa) domain, and recombinant GST pro- tein (rGST) were prepared from the transformed E. coli. BL21 containing the expression plasmid (pGEX-4T-3) with or without the insertion of human basigin cDNA. The human basigin domain of GST-basigin fusion protein com- prises 248 amino acid residues from position 22 to 269 on the human basigin protein sequence. Purification of the GST-basigin fusion protein was performed by glutathione- Sepharose 4B column chromatography. Similarly, a series of MBP-basigin fusion proteins and recombinant MBP (rMBP) were also prepared by using Protein Fusion & Puri- fication System (New England BioLabs). These MBP-basigin fusion proteins respectively contain a specified human basigin domain such as the full-length domain, the extra- cellular domain, the extracellular Ig-like domain I, the extra- cellular Ig-like domain II, the transmembrane-cytoplasmic domain and the cytoplasmic domain. Details of the human basigin domains are as follows. The full-length domain, 248 amino acid residues from amino acid position 22 to 269 on human basigin protein sequence; the extracellular domain, Fig. 1. Diagrammatic structure of human basigin. Basigin consists 176 residues from position 25 to 200; the extracellular Ig- of an extracellular domain, a transmembrane domain, a cytoplasmic like domain I, 77 residues from position 25 to 101; the domain and a signal peptide. The extracellular domain contains Ig- extracellular Ig-like domain II, 95 residues from position like domain I and Ig-like domain II. Numbers indicate the positions 106 to 200; the transmembrane-cytoplasmic domain, 67 of amino acid residues. Bold numbers indicate the limits of recombi- residues from position 203 to 269 and the cytoplasmic nant domains that construct the MBP-basigin fusion proteins. domain, 41 residues from position 229 to 269 (Fig. 1) [21]. The E. coli. TB1 containing the expression plasmid (pMAL- c2) with the insertion of a specified human basigin cDNA in Freund’s incomplete adjuvant. At 3 days after the final were used for production of MBP-basigin fusion proteins. booster injection, the mice were sacrificed and the spleens Purification of these MBP-basigin fusion proteins was per- were removed. Hybridomas were produced by the cell formed by amylose resin column chromatography. Purified fusion of P3U1mouse myeloid cells with spleen cells from products of GST-basigin fusion protein and MBP-basigin the immunized mice [33]. The cloning of hybridomas was fusion protein were found to have the expected molecular accomplished by three times limiting dilution procedure. weight by SDS polyacrylamide gel electrophoresis (SDS- Basigin-specific clones were selected by positive result to PAGE). the GST-basigin fusion protein and negative result to rGST on enzyme immune assay (EIA) for culture-supernatant of Antibody production hybridoma clones. Selected hybridoma clones were respec- BALB/c mice were subcutaneously immunized with tively inoculated into BALB/c mice by intraperitoneal (i.p.) the purified GST-basigin fusion protein emulsified in injection. At 18 days after inoculation, ascites containing Freund’s complete adjuvant. These mice were then boosted specific antibodies were respectively harvested from mice four times, every 2 weeks with the same antigen emulsified and then centrifuged at 1000Pg to remove the solid fraction. Distribution of Basigin by a Novel Antibody 137

IgG isotype of monoclonal antibodies against basigin from on glass slides by a centrifuge, Cytospin II (Shandon), and ascites were purified by Protein A-Sepharose CL-4B then were fixed in various media such as 10% buffered (Pharmacia) column chromatography. formalin and 95% methanol. L cells and Bsg
-L cells were respectively cultured on glass slides (LabTech Chamber, Cell preparation Nalgene Nunc International) for 3 days, and were then fixed L cells (mouse fibroblast like cells, strain L, purchased with 95% methanol. Cell samples for western blot analysis from The Health Science Research Resources Bank) [1] and were twice washed with phosphate buffered saline (PBS, L cell transfectants (Bsg
-L cells) containing the expression pH 7.4), and then dissolved in 2% SDS solution containing plasmid, pcDNA3.1() with human basigin cDNA [21] 10% glycerin. insertion, were prepared to confirm the specificity of novel antibodies against human basigin. The transfection of L Tissue preparation cells with expression plasmid was accomplished by calcium Human biopsy and autopsy tissue samples were fixed phosphate transfection procedure. These cells were main- with 10% buffered formalin and were then embedded with tained with Eagle’s MEM medium containing 10% fetal paraffin by routine procedure including ethanol dehydration bovine serum (FBS). Tumor cells such as KATO-III and xylene penetration. These paraffin embedded tissues (human gastric cancer cells) [28], HL-60 (human promyelo- were sectioned with a microtome and sections were mounted cytic leukemia cells) [9], Molt-4 (human T leukemia cells) on organosilane-coating glass slides. These paraffin speci- [25], Raji (human Burkitt’s lymphoma cells) [24] and THP-1 mens were used for immunohistochemical evaluation. (human monocytic leukemia cells) [32] were maintained with RPMI-1640 medium containing 10% FBS. These cul- Immunostaining tured cell lines were used as cell materials for immuno- Immunohistochemical and immunocytochemical stain- cytochemical evaluation and western blot analysis. For ing were performed by using biotinylated second antibody immunocytochemistry, tumor cell samples were mounted and peroxidase-streptoavidine complex (Histfine SAB kit,

Fig. 2. Detection of human basigin on western blot analysis by J-BIT-1. A: several types of cultured tumor cells (4P105 cells per lane) and GST-basigin fusion protein (250 ng per lane), which dissolved in 2% SDS solution with 2-mercaptoethanol (2-ME), were applied to SDS- PAGE, and were then blotted on PVDF membrane. The blotted membrane was applied to immunostaining with J-BIT-1. Each lane is as follows: lane 1) molecular weight (kDa) marker; lane 2) GST-basigin fusion protein; lane 3) THP-1 cell lysate; lane 4) KATO-III cell lysate; lane 5) Raji cell lysate; 6) HL-60 cell lysate; lane 7) Molt-4 cell lysate. GST-basigin fusion protein (53 kDa) and their fragments appear as brown bands on blotted membrane and are positive for J-BIT-1. Several J-BIT-1 antigens, which appear as brown broad bands containing two major bands are observed in cell lysates from THP-1, KATO-III, Raji and HL-60. The Molt-4 cell lysate is completely negative for J-BIT-1. B: L cells and Bsg+-L cells (transfectants) were dissolved in 2% SDS solution with or without 2-ME, and these lysates were applied to western blotting with J-BIT-1. Each lane is as follows: 1) Bsg+-L cell lysate with 2-ME; 2) Bsg+-L cell lysate without 2-ME; 3) L cell lysate with 2-ME; 4) L cell lysate without 2-ME. J-BIT-1 specifically stains the band (43 kDa) from Bsg+-L cells, but not from L cells. 138 Hiraishi et al.

NICHIREI). The immunostaining with a novel anti-basigin Tris-HCl buffer (pH 7.6). Specimens were finally counter- antibody was partially preceded by the microwave antigen stained with Mayer’s haematoxylin, and were enclosed retrieval procedure for formalin-fixed tissue specimens [31]. with cover slips. All procedures for immunostaining were Deparaffinized tissue specimens and fixed cell specimens performed at room temperature. Negative controls for were treated with 3% H2O2 solution. Following washes with immunohistochemical staining were performed by similar distilled water, formalin fixed specimens were placed in procedures without first antibody incubation. Non-immune plastic jars filled with 10 mM citrate buffer (pH 6.0), and the mouse IgG was also applied to cell immunostaining as jars were heated in a microwave oven (500 W) for 5 min. negative controls. The tissue specimens and fixed cell specimens were washed with distilled water, and rinsed in PBS (pH 7.4). The speci- Western blot analyses mens were incubated with 10% normal rabbit serum for Cell lysate containing 2% SDS from various cultured 30 min, and then incubated with anti-basigin mouse mono- cell lines were prepared for western blot analyses. These clonal antibody (10 g/ml) or non-immune mouse IgG samples were separated by SDS-PAGE with or without re- (purified myeroma IgG, 10 g/ml, Cappel) as the first anti- duction. A gradient polyacrylamide gel (4 to 20%, DAIICHI body for 60 min. After washes with PBS, specimens were PURE CHEMICALS) was used for SDS-PAGE. Separate incubated with biotinylated anti-mouse Ig antibody for 30 samples on the gel were then blotted on PVDF membrane min and were then washed again with PBS. In the next stage, (MILLIPORE) by semi-dry electroblotting. The membrane the specimens were incubated with peroxidase-streptoavi- was then immunostained with a novel anti-basigin mouse dine complex reagent for 30 min, then washed with PBS, monoclonal antibody. The detection of blotted antigens was and immersed with DAB/H2O2 solution for 5 to10 min. The performed by the SAB method similar to immunohisto- DAB/H2O2 solution for chromogen contained 0.02 mg/ml staining procedure except that H2O2 treatment and antigen 3,3'-diaminobenzidin (DAB) and 0.003% H2O2 in 0.05 M retrieval procedure was excluded. DAB or 4-chloro-1-

Fig. 3. Western blot analysis of J-BIT-1 reactivity against MBP-basigin fusion proteins. A series of MBP-basigin fusion proteins were applied to SDS-PAGE with reduction and were then blotted on PVDF membrane. The blotted membrane was applied to immunostaining with J-BIT-1 (B) and amide black staining (A and C). Lanes on figure A and B are as follows: lane 1) MBP (43 kDa); lane 2) MBP-basigin (full length) fusion protein (70 kDa); lane 3) MBP-basigin (extracellular domain) fusion protein (62 kDa); lane 4) MBP-basigin (Ig-like domain I) fusion protein (52 kDa); lane 5) MBP-basigin (Ig-like domain II) fusion protein (54 kDa); lane 6) MBP-basigin (transmembrane and cytoplasmic domain) fusion protein (50 kDa); lane 7) MBP-basigin (cytoplasmic domain) fusion protein (48 kDa). Figure C represents a lane of molecular weight (kDa) marker. Top major band of each lane on figure A and B appears at the expected position on SDS-PAGE. J-BIT-1 reacts to MBP-basigin fusion proteins containing the extracellular Ig-like domain II, such as lane 2, lane 3 and lane 5. Distribution of Basigin by a Novel Antibody 139 naphthol were used as the chromogen for peroxidase color- Characterization and specificity of the novel anti-basigin ing reaction. antibody On western blot analysis, J-BIT-1 clearly stained bands III. Results of the GST-basigin fusion protein and substances from several types of tumor cell line (Fig. 2A). GST-basigin Development of the mouse monoclonal antibodies against fusion protein was observed as major stained band at the human basigin expected position (53 kDa) on blotted membrane (Fig. 2A, As a result of the hybridoma cloning and selection, we lane 2). This means that the purified GST-basigin fusion obtained eight hybridoma clones, which were positively re- protein, the immunized recombinant basigin antigen was active to the GST-basigin protein, but not to rGST protein on strongly positive for J-BIT-1. Cell lysates from THP-1 cells, EIA by using the culture-supernatant of hybridoma clones. KATO-III cells, HL-60 cells and Raji cells were also posi- These clones were separately inoculated into BALB/c mice tive for J-BIT-1 on western blot analysis (Fig. 2A, lane 3, by i.p. injection. From these murine ascites, four IgG mono- lane 4, lane 5 and lane 6). These tumor cell lines have several clonal antibodies and four monoclonal IgM antibodies J-BIT-1 antigens containing two major bands, which differ were isolated. The antibodies were tested for their immuno- with the respect to size of their apparent molecular weights. histochemical reactivity against formalin-fixed, paraffin- These native antigens, which were expressed on tumor cells, embedded human tissues. Only one antibody, clone 53C366 were detected as typical broad bands on western blotting. appeared positive for immunohistochemical staining. We However, Molt-4 cell lysate was completely negative for selected this antibody, and named it J-BIT-1 (clone 53C366) J-BIT-1 (Fig. 2A, lane 7). Western blot analysis by using as the novel anti-basigin monoclonal antibody which ap- L cells and Bsg
-L cells could more directly indicate that peared to be useful for immunohistochemical staining. J-BIT-1 recognizes human basigin. The lysate from Bsg
-L cells which expressed human basigin (43 kDa) was positive

Fig. 4. Immunocytochemical staining of human basigin on cultured-tumor cells. Cell specimens, which were mounted on glass slides, were fixed in various media such as 95% methanol and 10% buffered formalin. Specimens were then immunocytochemically stained with J-BIT-1. Methanol-fixed Kato-III cells (A) are strongly stained with J-BIT-1. The positive signal, which is indicated by brown coloring, is observed on cell surface. The doubly fixed cells (C), which fixed with 10% formalin and 95% methanol, are also stained with J-BIT-1. Formalin-fixed Kato-III cells (B) and methanol-fixed Molt-4 cells (D) are negative for J-BIT-1 (D). Bar25 m (P640). 140 Hiraishi et al.

Fig. 5. Immunocytochemical staining of human basigin on Bsg+-L cells. L cells and Bsg+-L cells were cultured on glass slides for 3 days. Then, the cultured cells were fixed with 95% methanol and immunostained with J-BIT-1. Positive signals are observed in Bsg+-L cells only (A). L cells (without transfection) are completely negative for immunostaining with J-BIT-1 (B). Bar25 m (P320). for J-BIT-1, in contrast, the lysate from L cells was negative were completely negative (Fig. 5). These immunocytochem- (Fig. 2B). Moreover, the MBP-basigin fusion proteins con- ical findings were consistent with the finding of western blot taining the extracellular domain and the extracellular Ig-like analyses. domain II were clearly positive for J-BIT-1, but those containing the extracellular Ig-like domain I, the trans- Immunohistochemical distribution of basigin in human membrane-cytoplasmic domain, the cytoplasmic domain and tissues rMBP alone were completely negative (Fig. 3). These results In order to map out the distribution of basigin in various reveal that J-BIT-1 recognizes the extracellular Ig-like adult human organs, immunohistochemical staining with domain II of human basigin. J-BIT-1 was carried out. The immunostaining with J-BIT-1 Simple examinations were performed to search the op- revealed that basigin is widely expressed in various organs. timal fixation conditions for immunostaining with J-BIT-1. In the brain, J-BIT-1 stained blood capillaries that have rela- KATO-III cells were fixed with various fixation media, and tions with BBB (Fig. 6A). In the heart, cell surface of cardiac were then stained with J-BIT-1. KATO-III cells, which had muscle, especially sites of intercalated discs (junctures of been fixed with 95% methanol, were strongly positive for J- myocytes) were strongly positive for J-BIT-1 (Fig. 6B). In BIT-1 (Fig. 4A) while formalin-fixed KATO-III cells were the kidney, proximal convoluted tubules in the cortex, espe- almost negative for J-BIT-1 (Fig. 4B). The cells, which were cially at the basement membrane and cell-cell junctures doubly fixed with 10% formalin and then 95% methanol, were strongly positive for J-BIT-1 antibody (Fig. 6C). The were positive for J-BIT-1 (Fig. 4C). Other tumor cells fixed cell surface of liver cells also appeared positive for J-BIT-1 with 95% methanol, such as THP-1 cells, HL-60 cells and (Fig. 6D). In the gastroenteric tissues, surface epithelial cells Raji cells were also positive for J-BIT-1 (data not shown). in the intestinal mucosa and parietal cells in the mucosa were The positive signal of J-BIT-1 immunostaining was located positive for J-BIT-1 (Fig. 6E). In other tissues and organs, J- on the surface of methanol-fixed cells. Similar to western BIT-1 specifically reacted to the limited cell types, such as blot analysis, methanol-fixed Molt-4 cells were negative for trophoblasts of the placenta (Fig. 6F), basal cells of the strat- J-BIT-1 (Fig. 4D). Bsg
-L cells which had been fixed with ified squamous epithelium (Fig. 6G). Tissues found to be 95% methanol were also positive for J-BIT-1, but L cells negative for J-BIT-1 included glomerlus of the kidney (Fig.

Fig. 6. Typical immunohistochemical distribution of basigin on human tissues. Formalin-fixed, paraffin-embedded autopsy samples were applied to the immunostaining with J-BIT-1. Positive signals are indicated with brown coloring. Figures are as follows: (A) capillaries of the cerebrum (P160), (B) myocytes of the heart (P320), (C) the kidney (P160), (D) the liver (P320), (E) the gastric mucosa (P160), (F) trophoblast of the placenta (P320), (G) stratified squamous epithelium of the esophagus (P160), (H) the pulmonary alveoli (P160). Bars25 m. Distribution of Basigin by a Novel Antibody 141

Fig. 6 142 Hiraishi et al.

Fig. 7. Typical J-BIT-1 immunostaining on formalin-fixed specimens with the microwave antigen retrieval procedure. Formalin-fixed speci- mens with the microwave antigen retrieval procedure were applied to the immunostaining with J-BIT-1. Strong positive signals are observed on formalin-fixed specimens. Figures are as follows: (A) the liver (P320), (B) the kidney (P160). Bars25 m.

6C), endothelial cells of the blood vessels except the capil- IV. Discussion laries of the central nerve system, epithelium of lung alveoli (Fig. 6H), follicular cells of the thyroid and so on (partially In the present studies, we used two types of recom- data not shown). The microwave antigen retrieval procedure binant basigin proteins, such as the GST-basigin fusion for formalin-fixed tissue specimens could strengthen the protein and MBP-basigin fusion proteins to make the speci- positive signal of immunostaining with J-BIT-1 (Fig. 7). ficity of J-BIT-1 certain. J-BIT-1 could react to both GST- basigin fusion protein and MBP-basigin (full-length) fusion protein. The results of western blot analysis by using a series of MBP-basigin fusion proteins (Fig. 3B) indicate that J- Distribution of Basigin by a Novel Antibody 143

BIT-1 recognizes the extracellular Ig-like domain II of well known that blood capillaries of the brain are involved in human basigin. Moreover, this antibody could specifically BBB [26]. Proximal convoluted tubules of the kidney have recognize human basigin expressed in L cell transfectants an important role in selective reabsorption of threshold sub- as revealed by western blot analysis (Fig. 2B) and immuno- stances, such as glucose, amino acids, sodium chloride and cytochemical staining (Fig. 4). These results revealed that so on. The expression of basigin was located on the cell sur- J-BIT-1 is a specific antibody to human basigin. face, especially the site of cell-cell junctures and cell-base- Human basigin cDNA was cloned from a cDNA library ment membrane junctures. Recently it was reported that that was constructed from mRNA of KATO-III [21]. Of basigin (CD147 antigen) tightly associated with lactose course KATO-III cells naturally express human basigin, and transporters MCT1 and MCT4, but not MCT2, and the ex- were useful as positive controls for immunostaining with J- pression of MCT1 and basigin co-localize in the intercalated BIT-1. In the present studies, Molt-4 cells were used as the disk in myocytes [16]. Our observations in the heart tissues negative control for immunohistochemical evaluation with reveal that basigin is expressed on the cell surface of J-BIT-1, because the cells were completely negative for myocytes, especially on intercalated disks, junctures of 3 J-BIT-1 (Figs. 2A, 4D). Most tumor cells except for Molt-4 myocytes. It has been reported that  1 (VLA-3) and 6 cells had two major J-BIT-1 antigens different in apparent  1 (VLA-6) integrin, laminin/merosin receptors, form com- molecular weight on SDS-PAGE (Fig. 2A). Particularly plexes with basigin on HT1080 cell surface [3]. Further- native basigin antigens from KATO-III were detected as more, it was also reported that 2 types of CD147 antibodies typical broad bands containing two major bands on western could inhibit both the cell aggregation and the protein blotting. We believe that native basigin antigens on the cell tyrosine phosphorylation induced by CD98 (1 integrin) membrane are diversely glycosylated. In fact, human basigin ligation [6]. These reports suggest that basigin may be has potential asparagin glycosylation sites [21] (Fig. 1), and involved in integrin-associated cell adhesion or aggrega- mouse basigin is found as a carrier protein of the LTA tion. We cannot yet ensure whether expression of basigin receptor(s) [20]. Highly glycosylated J-BIT-1 antigens directly modulated cell adhesion and cell functions. We seemed to appear as the upper broad bands on SDS-PAGE. hypothesized that the expression of basigin on the cell Our results suggest that J-BIT-1 can recognize both recombi- membrane may be involved in cell-cell and cell to extra- nant basigin and native basigin, irrespective of whether it is cellular matrix interaction. glycosylated or not. Methanol-fixed KATO-III cells were In conclusion, we established a novel mouse mono- strongly positive for J-BIT-1 immunostaining (Fig. 4A). clonal antibody, named J-BIT-1, and demonstrated that J- Furthermore, 10% formalin-methanol doubly fixed KATO- BIT-1 antibody recognized human basigin. J-BIT-1 is III cells (Fig. 4C) and formalin-fixed paraffin-embedded appropriate to immunohistochemical staining for paraffin- tissue were positive for J-BIT-1 (Fig. 6A–G). However, embedded tissues and methanol-fixed cells. Basigin is widely formalin-fixed KATO-III cells unexpectedly were negative expressed in humans, and is located on the cell surface, (Fig. 3B). These results indicate that J-BIT-1 may recognize especially the site of cell adhesion. Our immunohistochemi- the site, which is sensitive to conformational changes with cal observations suggested that basigin might relate to basic alcohol-treatment. It is certain that J-BIT-1 is appropriate cell functions, such as selective permeation or barrier of for immunostaining of methanol-fixed cells and paraffin- permeation, which are promoted with specified cell-cell embedded tissues. J-BIT-1 immunostaining for paraffin- and cell-extracellular matrix interaction. embedded tissues with excessive formalin-fixation may need to precede by the microwave antigen retrieval proce- V. Acknowledgments dure. J-BIT-1 may be unsuitable for flow cytometric analy- sis using intact cells (non-fixed cells). We greatly appreciate Dr. Teruo Miyauchi for his kind Basigin has two separate Ig like domains (extracellular guidance and cooperation. Dr. Miyauchi retired from our Ig-like domain I and Ig-like domain II) and belongs to a company in the spring of 2001. Authors also greatly thank member of the Ig superfamily [20, 21]. Many Ig superfamily Dr. A. R. Saniabodi (Japan Immunoreserch Laboratories molecules, such as ICAM-1 (CD54 antigen), VCAM-1 Co., Ltd.) for editing our manuscript. (CD106 antigen) and NCAM have important roles in cell- cell interaction and cell adhesion [5]. We are particularly interested elucidating the essential role of basigin in cell VI. References function and pathogenesis. 1. Aleo, J. J., Orbison, J. L. and Hawkins, W. B. (1967) Histochem- Our immunohistochemical observations revealed that ical and biochemical studies of strain L fibroblasts treated with a basigin is widely expressed in various organs, but on specif- lathyrogen. 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