Advance Publication
Acta Histochem. Cytochem. 45 (4): 227–237, 2012
doi:10.1267/ahc.12008

1347-5800
Acta Histochemica
AHC
0044-5991
Japan
Tokyo,
Regular10.1267/ahc.12008
AHC12008
SocietyJapan
Articleof Histochemistry
et Cytochemica
and Cytochemistry

Immunohistochemical Examination of Novel Rat Monoclonal Antibodies against
Mouse and Human Podoplanin
Chiaki Kaji1,*, Yuta Tsujimoto2,*, Mika Kato Kaneko2, Yukinari Kato2 andYoshihiko Sawa1
1

Department of Morphological Biology, Fukuoka Dental College, 2–15–1 Tamura, Sawara-ku, Fukuoka 814–0193, Japan and
Molecular Tumor Marker Research Team, Yamagata University Global COE Program, Yamagata University Faculty of
Medicine, 2–2–2 Iida-nishi, Yamagata 990–9585, Japan

2

Received February 24, 2012; accepted June 14, 2012; published online July 25, 2012

© 2012
ThisThe
study
Japan
aims
Society
to developof Histochemistry
new monoclonal
and antibodies (mAbs) against mouse and human
podoplanin. Rats were immunized with synthetic peptides, corresponding to amino acids 38–
51 of mouse podoplanin or human podoplanin which is 100% homologous to the same siteof monkey podoplanin; anti-mouse podoplanin mAb PMab-1 (IgG2a) and anti-human mAb
NZ-1.2 (IgG2a) were established. In immunocytochemistry, the mouse melanoma B16-F10
and mouse podoplanin (mPDPN)-expressed CHO transfectant were stained by PMab-1;
human lymphatic endothelial cells (LEC) and human podoplanin (hPDPN)-expressedsquamous cell carcinoma HSC3 transfectant, were stained by NZ-1.2. Western-blot analysis
detected an about 40-kDa protein in CHO-mPDPN and B16-F10 by PMab-1, and in HSC3hPDPN and LEC by NZ-1.2. In frozen sections, PMab-1 reacted with mouse kidney, pulmonary
alveoli, pulmonary pleura, and salivary gland myoepithelial cells while NZ-1.2 reacted to thehuman salivary gland myoepithelial cells. The immunostaining of paraffin-embedded sections
also showed the reaction of PMab-1 or NZ-1.2 to the mouse or monkey kidney glomerulus,
pulmonary alveoli, and lung lymphatic vessels. These results indicate that the two novel rat
mAbs to the mouse and human/monkey podoplanin are useful for Western-blot andimmunostaining of somatic tissues on paraffin-embedded sections as well as frozen sections.
Key words: podoplanin, monoclonal antibody, PMab-1, NZ-1.2

I.

Introduction

Podoplanin, also termed Aggrus/gp36/E11 antigen, isa 38-kDa (mouse) or 36-kDa (human) mucin-type glycoprotein with a high content of sialic acid [7, 10]. Podoplanin was first identified as an antigen of the podocyte, the
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2
7
200?
;Received
45
2012
5accepted24.2.2012
14.6.2012*These authors contributed equally to this work.
Correspondence to: Yoshihiko Sawa, Department of Morphological
Biology, Fukuoka Dental College, 2–15–1 Tamura, Sawara-ku,
Fukuoka 814–0193, Japan. E-mail: ysawa@college.fdcnet.ac.jp
Yukinari Kato, Molecular Tumor Marker Research Team, Yamagata
University Global COE Program, Yamagata University, Faculty of
Medicine, 2–2–2 Iida-nishi, Yamagata 990–9585, Japan.
E-mail: yukinari-k@bea.hi-ho.ne.jp

kidney glomerular epithelial cell [1], and is also known asa marker of lymphatic endothelium and alveolar epithelium[22, 27, 35]. There has also been reports that podoplanin
is expressed in osteocytes and osteoblasts [33], mesothelial
cells [14], epidermal basal layer cells [27], tooth germepithelial cells, salivary gland myoepithelium [4–6, 20],
thymus type I epithelial cells, prostate myofibroblasts, follicular dendritic cells, and immature cells such as fetal germ
cells or developing Sertoli cells [17, 22, 27, 35, 36]. The
podoplanin promoter region lacks the TATA and CAT box,
but includes a CpG island with putative Sp1 and AP-4 sites[3]. Such promoters are usually present at the 5'-flanking
region of housekeeping genes, and CpG hypermethylation
has been linked with their transcriptional repression. Thereis, however, CpG methylation-dependent podoplanin transcription enhancement, which is probably based on chroma© 2012 The Japan Society of Histochemistry and Cytochemistry

228

Kaji et al.

tin structure modifications; therefore, the CpG methylation
may induce the specific expression in different kinds ofsomatic cells [3, 34].
The C-type lectin-like receptor 2 (CLEC-2) is the specific receptor for podoplanin, which mediates the platelet
aggregation without blood coagulation factors and plasmacomponents [12, 30]. Podoplanin consists of an extracellular domain rich in serine and threonine residues,
and with a transmembrane domain, and a cytoplasmic tailfor protein kinase C and cAMP phosphorylation [8].
Podoplanin has a platelet aggregation-stimulating (PLAG)
domain, of which only the Thr52 is O-glycosylated with
disialyl-core1 (NeuAc-α2-3Galβ1-3 (NeuAcα2-6) GalNAcα1O-Thr) is in an extracellular domain [9]. The binding of
podoplanin to CLEC-2 is dependent on the sialic acid of
O-glycan in the PLAG domain by the lectin activity,and mediates the platelet aggregation [12]. Three tandem
repeats of the PLAG domain are conserved in podoplaninhomologues of the rat, hamster, dog, bovine, human, andmouse [8]. The podoplanin homologues conserve theEDxxVTPG segment in the extracellular domains, which
are critical for the platelet aggregation-inducing activities.
To date, several anti-mouse/human podoplanin antibodies have been established. The D2-40 antibody which
recognizes the oncofetal antigen M2A of human podoplanin, and is useful in immunohistochemistry [2, 23]. Anotheranti-human podoplanin mAb (Clone AB3, AngioBio) possesses the very same characteristics as D2-40 [21]. The NZ1 antibody against the human PLAG domain suppresses
both podoplanin-CELC-2 interaction and podoplanininduced hematogenous pulmonary metastasis [11, 12, 21].
The 8F11 monoclonal antibody against mouse podoplanininhibits platelet aggregation and lung metastasis of mousecolon carcinomas [29, 31]; however, 8F11 has not beenreported to be useful in immunohistochemistry. Podoplaninexpressing tumor cells may survive by aggregating platelets
around the cells [12, 16], and podoplanin expression isassociated with a poor prognosis of some kinds of tumors,and therefore podoplanin could be valuable as an anticancer target [13, 18, 28, 37]. Especially, the developmentof antibodies, which recognize the PLAG domain strongly,
may contribute to anti-metastasis therapies against podoplanin-positive tumors. A number of anti-mouse/human
podoplanin antibodies have been reported, however novelanti-podoplanin antibodies are still required to examine
the precise distribution of podoplanin in somatic tissue.
This study aims to develop new antibodies for the PLAG
domain of mouse and human podoplanin, and investigatesthe specificity for podoplanin of new antibodies in themouse, monkey, and human tissue.

II. Materials and Methods
Subjects and cultured cells

Human tissue of the sublingual glands of five individuals, 60–80 years of age, was obtained immediately
following the body donation to Fukuoka Dental College foreducation and research of oral medicine. All procedures
were performed with appropriate informed consent according to the protocol of the human gift registry programs of
Fukuoka Dental College. The study used C57BL/6N mice
(CLEA Japan, Inc., Tokyo, Japan) and Sprague-Dawley(SD) rats (CLEA) in the immunohistochemical examinationand in the development of rat antibodies, respectively.
The mouse myeloma P3U1 cells (CRL-1597) and Chinese
hamster ovary (CHO) K1 cells (CRL-2243) were obtained
from the American Type Culture Collection (ATCC,
Manassas, VA). The human tongue squamous cell carcinoma cell line (HSC3) was obtained from the JapaneseCollection of Research Bioresources Cell Bank (Osaka,
Japan). The P3U1 and CHO cells or HSC3 were cultured
in RPMI 1640 or Dulbecco’s Modified Eagle Medium(DMEM, Wako Pure Chemical Industries Ltd., Osaka,
Japan), respectively, including 2 mM L-glutamine (Wako)
supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Life Technologies Japan Ltd., Tokyo, Japan)
at 37°C in a humidified atmosphere of 5% CO2 and 95%
air. The neonatal human dermal lymphatic microvascularendothelial cells (LEC: HMVEC-dLy, CC-2812, Lonza,
Walkersville, MD) and mouse melanoma B16-F10 cells(CRL-6475, ATCC), which are known to express intrinsic
podoplanin [32], were also used as a positive control for
intrinsic podoplanin. The LEC and B16-F10 were cultured
in endothelial cell medium EGM-2MV supplemented with
5% FBS (Lonza) or DMEM (Wako), respectively.
Stable transfection

The CHO cells were transfected with appropriate
amounts of plasmid: pcDNA3-mouse podoplanin-DYKDDDDK (FLAG) [10] using LipofectAMINE 2000 reagent(Life Technologies Japan Ltd.) according to the manufacturer’s instructions. The HSC3 cells were transfectedwith a plasmid: pcDNA3-human podoplanin-DYKDDDDK(FLAG) [10] by electroporation using CUY21Pro-Vitro(NepaGene Co., Ltd., Ichikawa, Japan) according to theoriginal protocol. Briefly, the confluent monolayer of HSC3
cells in 6-well plates were trypsinized, harvested, andsuspended in the Opti-MEM I (Life Technologies Japan
Ltd.). A 10-μl volume of the RNase and DNase freedeionized distilled water containing 2 μg of pcDNA3human podoplanin was added into a 90-μl volume of the
HSC3 cell suspension (1.0×106) in the electroporation
cuvettes (NepaGene). Electroporation was repeated 10
times: 125 V for 10 ms, offtime for 50 ms, 20 V for 50 ms,
and off time for 50 ms. After the electroporation, thecell suspension was immediately disseminated in DMEM
with FBS, and the stable transfectants of CHO-mouse
podoplanin (CHO-mPDPN) and HSC3-human podoplanin(HSC3-hPDPN) were selected by cultivating in a medium
containing 0.5 mg/ml of Geneticin (Wako Pure Chemical
Industries Ltd.). Mouse and human podoplanin genes in
pcDNA3 were confirmed by sequence analysis (Macrogen
Japan, Tokyo, Japan).

Production of Anti-Podoplanin Antibodies

production of anti-mouse podoplanin and anti-human
podoplanin monoclonal antibodies

The SD rats were immunized by neck subcutaneous injections of the synthetic peptide GDGMVPPGIEDKIT(mpp3851) or EGGVAMPGAEDDVV (hpp3851), corresponding to amino acids 38–51 of mouse podoplanin or 38–
51 of human podoplanin, plus C-terminus cysteine conjugated with keyhole limpet hemocyanin (KLH) with alum
adjuvant (Imject Alum, Thermo Scientific Inc., Rockford,
IL). The amino acids 38–51 of human podoplanin are
100% homologous to the amino acids 38–51 of monkey
podoplanin. One week after the immunization, a secondary
immunization was performed by an intraperitoneal booster
injection 2 days before spleen cells were harvested. The
spleen cells were fused with P3U1 cells using a Sendai
virus (Hemagglutinating Virus of Japan: HVJ) envelope:
GenomONE-CF (Ishihara Sangyo Kaisha, Ltd., Osaka,
Japan) according to the manufacturer’s instructions. The
hybridomas were grown in RPMI medium with hypoxanthine, aminopterin and thymidine selection medium supplement (Invitrogen Corp., Carlsbad, CA). The culture
supernatants were screened by enzyme-linked immunosorbent assay (ELISA) for binding to the synthetic peptide.
The synthetic peptide GDGMVPPGIEDKIT (mpp3851)
or EGGVAMPGAEDDVV (hpp3851), corresponding toamino acids 38–51 of mouse podoplanin or 38–51 ofhuman podoplanin were immobilized on Maxisorp 96-wellimmunoplates (Thermo Fisher Scientific Inc.) at 1 μg/ml
for 30 min, respectively. After blocking with 1% BSA in
PBS, the plates were incubated with culture supernatant,followed by 1:1,000 diluted peroxidase-conjugated anti-rat
IgG (Dako, Glostrup, Denmark). The enzymatic reaction
was conducted with a substrate solution containing 3,3',5,5'tetramethylbenzidine (Thermo Fisher Scientific Inc.). The
optical density was measured at 655 nm with a Benchmark
microplate reader (Bio-Rad Laboratories Inc., Philadelphia,
PA). These reactions were performed with a volume of 50
μl at 37°C. The Hybridoma was further cultivated in CD
Hybridoma medium (Life Technologies Japan Ltd.). The
culture mediums were centrifuged, and the obtained supernatant was applied to a column of protein G-Sepharose(Thermo Scientific Inc., Rockford, IL).
ImmunohistochemistryThis study used tissue of kidneys, lungs, tongues, andsublingual glands of eight-week-old male C57BL/6N mice(n=5) (CLEA). The tissue was collected after euthanasia
by intraperitoneal injection with sodium pentobarbital (10ml/kg, Nembutal, Abbott Laboratories, North Chicago, IL).
The protocol for the animal use was reviewed and approvedby the animal experiment committee of Fukuoka Dental
College in accordance with the principles of the Helsinki
Declaration. Frozen 10 μm sections of human sublingual
glands from donated human bodies and mouse tissue were
cut in a cryostat, and fixed in 100% methanol for 10 min at–20°C. The sections were treated with 0.1% goat serum for
30 min at 20°C, and then the sections were treated with 0.5229μg/ml of antibodies: monoclonal rat anti-mouse podoplanin(PMab-1), monoclonal hamster anti-mouse podoplanin(Mab-8.1.1, AngioBio Co., Del Mar, CA), monoclonal rat
anti-human podoplanin (NZ-1.2), monoclonal mouse antihuman podoplanin (AB3, AngioBio), polyclonal rabbit
anti-mouse α-smooth muscle actin (α-SMA) (Epitomics,
Burlingame, CA), and polyclonal rabbit anti-FLAG antibody, which recognizes DYKDDDDK (Sigma-Aldrich,
St. Louis, MO) for 8 hr at 4°C. After the treatment withprimary antibodies, the sections were immunostained for
0.5 hr at 20°C with 0.1 μg/ml of second antibodies: Alexa
Fluor (AF) 488 or 568-conjugated goat anti-mouse, goatanti-hamster, goat anti-rabbit, or goat anti-rat IgGs (LifeTechnologies Japan Ltd.). The nuclei were counterstainedwith DAPI (Sigma-Aldrich). The immunostained sections
were mounted in 50% polyvinylpyrrolidone solution, andexamined by fluorescence microscopy BZ-8100 (Keyence
Corp., Osaka, Japan) or laser-scanning microscopy (LSM
710, Carl Zeiss, Jena, Germany) with a PlanApochromatic
60× oil immersion objective lens (numerical aperture ×1.3).
The CHO, CHO-mPDPN, HSC-3, HSC3-hPDPN, B16-F10,
and LEC were cultured on cover slips in 6-well plates.
The confluent monolayer of the cells on the cover slips
was immersed in 4% paraformaldehyde-PBS for 5 min atroom temperature. After the treatment with 0.1% goat
serum for 30 min, the cells were immunostained as described above. The cover slips with the cells were mounted
in 50% polyvinylpyrrolidone solution on slide glasses, andexamined by laser-scanning microscopy. In the assessmentof PMab-1 and NZ-1.2 about the specificities for mouseand human podoplanin, CHO-mPDPN and HSC3-hPDPN
cells which are the transfectants of podoplanin gene, and
B16-F10 and LEC cells expressing wild type podoplanin,
were used as positive controls for reactivities to podoplanin on cultured cells while CHO and HSC3 were usedas negative controls. The Mab-8.1.1 or AB3-positive kidney
podocytes, lung epithelial cells, lymphatic vessels, andsalivary gland myoepithelial cells were used as positive
controls for reactivities to podoplanin on somatic tissue
while the connective tissue, muscle cells, and blood vessels
in tissue were used as negative controls.
To make paraffin-embedded sections, perfusion fixation was administered with 500 ml of fixative solution
containing 4% formaldehyde-PBS for the eight-week-old
male C57BL/6N mice after euthanasia by intraperitoneal
injections with sodium pentobarbital. The dissected tissue
was additionally fixed by immersion in a 10% formalinPBS solution, with a volume 50 times larger than theimmersed tissue, for 8 hr at room temperature as fixing of
the tissue for longer than 24 hr leads to masking or
destruction of the podoplanin antigen. For the collection
from the lungs, the fixative solution was injected into theeuthanized mouse lungs by a syringe through the oral cavity
to prevent the lung from collapsing by atmospheric pressure
at the thoracotomy on the perfusion fixation. The 5-μm
thick paraffin-embedded sections were cut using a rotary
microtome, mounted onto 3-aminopropyltriethoxysilane-230

Kaji et al.

coated slides, rehydrated in xylene and ethanol, and rinsed
with deionized distilled water for 10 min. Slides were
immersed in 10 mM sodium citrate buffer (pH 6.0) andboiled in a microwave oven for 10 min to break formalinformed protein cross-links masking the podoplanin antigen.
After the boiling, the sections were rinsed by Phosphatebuffered saline (PBS) and blocked with 0.1% goat serum.
The immunostaining was performed using the first andsecond antibodies diluted into the blocking solution. Allprocedures were performed after washing with PBS for 30
min at room temperature. Also, the paraffin-embedded
sections of monkey kidneys and lungs (HistoMap, Wako)
were used to investigate the specificity of NZ-1.2 for thehuman podoplanin.
Reverse transcription (RT)-PCR

The total RNA extraction from CHO, CHO-mPDPN,
HSC-3, HSC3-hPDPN, B16-F10, and LEC was achieved
with a QIAshredder column and an RNeasy kit (Qiagen,Inc., Tokyo, Japan). Contaminated genomic DNA wasremoved using DNAfree (Ambion, Huntingdon, UK), andthe RT was performed on 30 ng of total RNA, followed
by 25–30 cycles of PCR for amplification using the Ex
Taq hot start version (Takara Bio Inc., Otsu, Japan) with 50
pM of primer sets: mouse β-actin (forward, 5'-GTTCTAC
AAATGTGGCTGAGGA; reverse, 5'-ATTGGTCTCAAG
TCAGTGTACAG, 411 bp), mouse podoplanin coding sequence region (forward, 5'-CACCTCAGCAACCTCAGAC;

reverse, 5'-AAGACGCCAACTATGATTCCAA, 192 bp),
FLAG-tagged mouse podoplanin (forward, 5'-CACCTCA
GCAACCTCAGAC; reverse, 5'-TGTCGTCATCGTCTTT
GTAGTC, 284 bp), human β-actin (forward, 5'-ATGTTTGAGACCTTCAACAC; reverse, 5'-CACGTCACACTTCATGATGG, 495 bp), human podoplanin (forward, 5'-TGACAACTCTGGTGGCAACAAG; reverse, 5'-GGTCACTG
TTGACAAACCATCTTT, 200 bp), and FLAG-tagged
human podoplanin (forward, 5'-TGACAACTCTGGTGGCAACAAG; reverse, 5'-CGTCTTTGTAGTCGGGCGAG,
309 bp) where the specificities had been confirmed by the
manufacturer (Sigma-Genosys Ltd., Cambridge, UK). The
correct size of the amplified RT-PCR products were confirmed by electrophoresis on 2% agarose gel (NuSieve;
FMC, Rockland, ME, USA) and visualized by Syber Green(Takara).
Western-blot analysis

The CHO, CHO-mPDPN, HSC-3, HSC3-hPDPN,
B16-F10, and LEC cells forming a confluent monolayer inthe 6-well plate were harvested by a cell scraper, andsolubilized in 100 μl of cell lysis buffer (Cell Signaling
Technology, Inc., Danvers, MA). The lysate of whole cell
protein (1 mg/ml) was mixed with an equal volume ofsample buffer (Bio-Rad Laboratories Inc., Hercules, CA).
A 20-μl volume of sample was loaded on 15% polyacrylamide gel by electrophoresis with Mini-protean III (BioRad), and the separated proteins were transferred onto a

Fig. 1. (A) Immunostaining for podoplanin and FLAG on cultured cells. The CHO and B16-F10 cells were exposed to a cocktail of PMab-1(green) and anti-FLAG (red) (PMab-1-FLAG). The HSC3 and LEC cells were exposed to a cocktail of NZ-1.2 (green) and anti-FLAG (NZ-1.2FLAG). The nuclei were counterstained with DAPI. The CHO was not stained by PMab-1 or anti-FLAG. The B16-F10 was stained by PMab-1
at the cytoplasm but not by anti-FLAG. The HSC3 was not stained by NZ-1.2 or anti-FLAG but LEC was stained by NZ-1.2 at the cell-celljunction and the cytoplasm. (B) Immunostaining of the transfectants for mouse and human podoplanin-FLAG genes. The CHO-mPDPN cells
were exposed to a cocktail of PMab-1 (green) and anti-FLAG (red). The HSC3-hPDPN cells were exposed to a cocktail of NZ-1.2 (green) andanti-FLAG. The nuclei were counterstained using DAPI. The FLAG protein expressed on CHO-mPDPN and HSC3-hPDPN were stained byanti-FLAG. The mouse or human podoplanin expressed on CHO-mPDPN or HSC3-hPDPN were stained by PMab-1 or NZ-1.2. The merged
images indicate that the PMab-1 or NZ-1.2-reacting region on CHO-mPDPN or HSC3-hPDPN coincided with the anti-FLAG reacting region.
Nuclei were counterstained using DAPI. Bar=50 μm.

Production of Anti-Podoplanin Antibodies

PVDF membrane (Life Technologies Japan Ltd.). After
blocking of the PVDF membrane by 5% skim milk for 3 hr
at 20°C, the membrane was treated with the blocking agent
containing PMab-1 and NZ-1.2 (10 μg/ml) for 2 hr at 20°C,
and with peroxidase-conjugated goat anti-rat (1 μg/ml)
for 1 hr at 20°C, and then visualized by the substrate ofa Vector Elite ABC kit and a Vector VIP kit (Vector
Laboratories, Burlingame, CA) or Immun-Star WesternC
Chemiluminescence kit (Bio-Rad), and scanned by animage analyzer LAS-3000 mini (Fujifilm Corp., Tokyo,
Japan).

III. Results
Examination of anti-mouse podoplanin monoclonal antibody
PMab-1 and anti-human podoplanin monoclonal antibody
NZ-1.2 against cultured cells

The hybridoma clones producing anti-mouse podoplanin monoclonal antibody PMab-1 (IgG2a subclass) or antihuman monoclonal antibody NZ-1.2 (IgG2a subclass) were
established. In the immunocytochemical analysis on cul-

231

tured cells, CHO was not immunostained by PMab-1 oranti-FLAG, whereas B16-F10 was stained by PMab-1(Fig. 1). The HSC3 was not immunostained by NZ-1.2 oranti-FLAG, while LEC was stained by NZ-1.2 at the cellcell junction and the cytoplasm. The CHO-mPDPN, whichis the stable transfectant of CHO-K1 with FLAG-taggedmouse podoplanin gene, was stained by PMab-1 and antiFLAG (Fig. 1). The HSC3-hPDPN, which is the stabletransfectant of HSC3 with the FLAG-tagged human
podoplanin gene, was stained by NZ-1.2 and anti-FLAG.
The stained region by PMab-1 or NZ-1.2 on CHO-mPDPN
or HSC3-hPDPN coincided with the anti-FLAG reaction.
The PCR products for the mouse podoplanin genecoding sequence (CDS) and the FLAG-tagged mouse
podoplanin gene were not detected in CHO, but detected in
CHO-mPDPN (Fig. 2). The PCR products of podoplanin
CDS were detected solely in the mouse melanoma B16-F10,
which expresses intrinsic podoplanin. Western-blot analysis
showed that PMab-1 did not react to the whole cell proteinfrom CHO or HSC3-hPDPN, whereas about 40-kDa protein
was detected in CHO-mPDPN and B16-F10 (Fig. 2). The

Fig. 2. Gene and protein analysis for podoplanin on cultured cells. (A) RT-PCR analysis on the mouse podoplanin mRNA expression. The PCR
products of mouse podoplanin gene coding sequence (cds) and the FLAG-tagged mouse podoplanin gene (flag) were not detected from theCHO, but were detected from CHO-mPDPN. The PCR products of podoplanin CDS were also detected from B16-F10. MW, molecularweight marker. (B) Western-blot analysis of the mouse podoplanin protein. The PMab-1 did not react to the whole cell protein from CHO or
HSC3-hPDPN, whereas about an 40-kDa protein was detected from CHO-mPDPN and B16-F10. The molecular weight of the FLAG-tagged
podoplanin in CHO-mPDPN is slightly larger than the intrinsic podoplanin of about 40 kDa in B16-F10 because of the molecular weight of theFLAG. MW, molecular weight marker. (C) RT-PCR analysis for the human podoplanin mRNA expression. The PCR products of the human
podoplanin coding sequence (cds) and the FLAG-tagged human podoplanin gene (flag) were not detected from HSC3, but were detected from
HSC3-hPDPN. The PCR products of podoplanin CDS were also detected from LEC. MW, molecular weight marker. (D) Western-blot analysisof the human podoplanin protein. The NZ-1.2 did not react to the whole cell protein from HSC3 or CHO-mPDPN, whereas an about 40-kDa
protein was detected from the HSC3-hPDPN and LEC. The molecular weight of the FLAG-tagged podoplanin from HSC3-hPDPN is slightly
larger than the intrinsic podoplanin of about 40 kDa from LEC. MW, molecular weight marker.

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Kaji et al.

molecular weight of FLAG-tagged podoplanin in CHOmPDPN is slightly larger than the intrinsic podoplanin in
B16-F10. The PCR products of human podoplanin CDS
and the FLAG-tagged human podoplanin gene were notdetected in HSC3, but detected in HSC3-hPDPN (Fig. 2).
The PCR products of human podoplanin CDS were detected
in LEC, which expresses intrinsic podoplanin. Western-blot
analysis showed that NZ-1.2 did not react with the wholecell protein from HSC3 or CHO-mPDPN, whereas about a
40-kDa protein was detected in HSC3-hPDPN and LEC(Fig. 2). The molecular weight of the FLAG-tagged human
podoplanin from HSC3-hPDPN is slightly larger than the
intrinsic podoplanin from LEC.
Immunohistochemical analysis of PMab-1 and NZ-1.2 on themouse and human tissues

In the immunostaining for podoplanin of the mouse
kidney, PMab-1-reacting region on the glomerular capillarycoincided with another anti-mouse podoplanin Mab-8.1.1reacting region in the glomerulus of the renal cortex withoutcross-reaction to the uriniferous tubules (Fig. 3). In the
immunostaining for podoplanin of the mouse lung, thePMab-1-reacting region on pulmonary alveoli and thepulmonary pleura coincided with the Mab-8.1.1-reacting
region, with a reaction stronger than PMab-1. Immunostaining for α-SMA and podoplanin of mouse tongue, theanti-α-SMA reacted to blood vessels, whereas the PMab-1
reacted to lymphatic vessels in the tunica muscularis(Fig. 4). In the mouse lingual gland, reaction productswith PMab-1 were observed on acini and on the duct, andon lymphatic vessels without cross-reaction to any tissue,
while anti-α-SMA reacted to the secretory terminal end
pieces of acini and on the duct with cross-reaction to intraacinar components and to lingual mucous epithelia (Fig. 4).
In the mouse sublingual gland, the terminal secretory end
pieces were densely composed of large mucous-filled cells(Fig. 5). The intercalated and striated ducts were composedof columnar cells with intensely acidopillic cytoplasm andwell developed when compared with the lingual gland. In
the immunostaining against podoplanin and α-SMA of themouse sublingual glands, the regions of myoepithelial cells
around acini and the basal side of intercalated and striatedducts reacted with anti-α-SMA, and Mab-8.1.1 or PMab-1.
The cell-cell junction of acinar cells and the luminal side ofintercalated ducts, where myoepithelial cells are absent,
were immunostained by anti-α-SMA, but not by Mab-8.1.1
or PMab-1. The Mab-8.1.1 reacted to almost the same
region as that stained by PMab-1, but also showed a weakcross-reaction with connective tissue around the striatedduct. In the human sublingual gland, the terminal secretory
end pieces of acini were densely composed of large
mucous-filled cells with a few serous pyramidal cells(Fig. 6). In the immunostaining for podoplanin and α-SMA
of human sublingual glands, the regions of myoepithelial
cells around mucous acini reacted by anti-α-SMA, antihuman podoplanin AB3, and NZ-1.2 while the basal portionof serous acini partially showed a weak reaction to theantibodies. The cell-cell junction of acinar cells and theluminal side of ducts, where myoepithelial cells wereabsent, were immunostained by anti-α-SMA, but not by

Fig. 3. Immunostaining for podoplanin on mouse kidney and lung tissue. In the glomerulus of the renal cortex, the merged image indicatesthat the PMab-1-reacting region on the glomerular capillary (GA) (green) coincides with the Mab-8.1.1-reacting region (red). The PMab-1 andMab-8.1.1 did not react to uriniferous tubules (UT). In the lung, the merged image indicates that the pulmonary pleura (PP) and a part of thepulmonary alveoli (PA) were immunostained by PMab-1 (green). The immunoreaction of Mab-8.1.1 (red) to alveolar cells was stronger thanPMab-1 and the Mab-8.1.1 specificity was broader comparing with PMab-1. Nuclei were counterstained using DAPI. Bar=20 μm.

Production of Anti-Podoplanin Antibodies

233

Fig. 4. Immunostaining for podoplanin on mouse radix linguae tissue. In the tunica muscularis, blood vessels were stained by anti-α-SMA(arrow) (red), whereas lymphatic vessels were stained by PMab-1 (arrowheads) (green). In the lingual gland reaction products with PMab-1(green) were also observed on acini (arrows) and on the duct (arrowhead), and on lymphatic vessels (asterisks) without cross reaction to any
tissue, while reaction products with anti-α-SMA (red) were observed on the secretory terminal end pieces of acini (arrows) and on the duct(arrowhead) with cross reaction to the intra-acinar components and to lingual mucous epithelia (asterisk). Nuclei were counterstained using
DAPI. Bar=100 μm.

Fig. 5. Immunostaining for podoplanin on mouse sublingual gland tissue. The H-E staining shows that the secretory terminal end pieces of acini
are composed of mucous-filled acinar cells. The intercalated duct (ID) is found interposed between acinar cells, and has a narrow lumen surrounded by cuboidal cells. The striated duct (SD) is composed of columnar cells with acidopillic cytoplasm. The regions of the myoepithelial
cells around acini and the basal side of the intercalated and striated ducts reacted by anti-α-SMA (red), anti-podoplanin Mab-8.1.1 (red), andPMab-1 (green). The cell-cell junction of the acinar cells where myoepithelial cells are not present were immunostained with anti-α-SMA(arrowheads) but not with Mab-8.1.1 or PMab-1. The Mab-8.1.1 reacted more strongly to the PMab-1-reacting region, but also showed a weakcross reaction with connective tissue around the striated duct (asterisk). Nuclei were counterstained by DAPI. Bar=100 μm.

234Kaji et al.

Fig. 6. Immunostaining for podoplanin on human sublingual gland tissue. The H-E staining shows the secretory terminal end pieces which
composed of large mucous-filled acinar cells, and a few serous pyramidal cells stained to a stronger extent than the mucous acini. The regionsof myoepithelial cells around mucous acini reacted by anti-α-SMA (arrows) (red), AB3 (arrows) (red), and NZ-1.2 (arrowheads) (green) while
the basal portion of serous acini adjacent to mucous acini partially showed a weak reaction to the antibodies. The cell-cell junctions of acinar
cells where myoepithelial cells are absent were also stained by anti-α-SMA (asterisk), but not with AB3 or NZ-1.2. The AB3 reacted to theNZ-1.2-staining region to a strikingly weaker extent and also showed a cross-reaction with nuclei. Nuclei were counterstained using DAPI.
Bar=100 μm.

Fig. 7. The immunostaining of the paraffin-embedded sections by PMab-1 and NZ-1.2. The PMab-1 reacted on the glomerulus (GA) of the renal
cortex without any cross reaction to uriniferous tubules (UT) in the mouse kidney, and reacted on the pulmonary alveoli (PA, arrowheads) andlymphatic vessels (arrows) in the mouse lungs. The NZ-1.2 reacted on the glomerulus (arrows) of the renal cortex and lymphatic vessels (arrowheads), but not on the blood vessels (B) in the monkey kidney, and reacted on the pulmonary alveoli (arrowheads) and lymphatic vessels(arrows) in the monkey lung. Bar=50 μm.

Production of Anti-Podoplanin Antibodies

AB3 or NZ-1.2. The AB3 reacted to the NZ-1.2-staining
region to a strikingly weaker extent; and also showed across-reaction with nuclei. In the immunostaining ofparaffin-embedded sections, PMab-1 reacted to the mouse
kidney glomerulus, and to the mouse pulmonary alveoli
and lung lymphatic vessels (Fig. 7). The NZ-1.2 also
reacted to the monkey glomerulus and kidney lymphatic
vessels, and to the monkey pulmonary alveoli and lung
lymphatic vessels.

IV.

Discussion

A rat monoclonal antibody NZ-1, which is specific tohuman podoplanin has previously been established [11],and NZ-1 has enabled neutralized human podoplanininduced platelet aggregation and pulmonary metastasis [12].
The NZ-1 antibody is also useful in immunohistochemistry,suggesting that the NZ-1 immunogen is very effective in
producing anti-podoplanin antibodies. However, NZ-1 does
not react with mouse, rat, or hamster podoplanin (data notshown). For analyzing mouse podoplanin expression invivo, the investigation reported here intended to produce anovel anti-mouse podoplanin by immunizing rats withamino acids 38–51 of mouse podoplanin, which corresponds to the NZ-1 immunogen. It was possible to establish
both an anti-mouse podoplanin antibody (PMab-1) and ananti-human podoplanin antibody (NZ-1.2), which are very
useful in immunohistochemistry. In the analysis using
cultured cells, CHO was not immunostained by PMab-1 oranti-FLAG; B16-F10 was stained by PMab-1; and CHOmPDPN was stained by PMab-1 and anti-FLAG, suggestingthat the reaction of PMab-1 is specific for the mouse
podoplanin-expressing cells (Fig. 1). We have previously
reported the gene and protein expression of podoplanin in
the cultured human lymphatic endothelial cell LEC [24–26]. The study here showed that LEC was stained by NZ1.2. Further, HSC3 was not immunostained by NZ-1.2 oranti-FLAG, whereas the PMab-1 or NZ-1.2-reacting region
on CHO-mPDPN or HSC3-hPDPN coincided with theanti-FLAG-reacting region, suggesting that the reaction ofPMab-1 or NZ-1.2 is specific for mouse or human podoplanin (Fig. 1). The specificity of PMab-1 or NZ-1.2 was also
confirmed by Western-blot analysis (Fig. 2). The mouse
podoplanin gene and protein were not detected from CHO,
but were detected from CHO-mPDPN and from mousemelanoma B16-F10 expressing intrinsic podoplanin. The
human podoplanin gene and protein were not detected from
HSC3, but was detected from HSC3-hPDPN and fromhuman lymphatic endothelial cell LEC expressing intrinsic
podoplanin.
Podoplanin is known as an antigen of podocytes ofkidney [1]. It is thought that podoplanin plays a role inmaintaining the shape of podocyte foot processes andglomerular permeability, because podocyte foot processes
are extensive flattenings, and podoplanin is selectively
reduced to <30% in a rat model of nephropathy by puromycin aminonucleoside nephrosis accompanying severe pro-235

teinuria [15]. Podoplanin is also well known as a lymphatic
endothelial cell marker, and is homologous to the T1α-2gene, which encodes the type I alveolar cell specific antigen[2, 23, 35]. Podoplanin−/− mice have defects in the lymphatic formation with congenital lymphedema, but not havedefects in the blood vessel pattern formation, and they die
at birth because of the respiratory failure [27]. In the
immunohistochemical study on podoplanin expression ofmouse kidneys here, the PMab-1-reacting region on theglomerular capillary coincided with the Mab-8.1.1-reacting
region in the glomerulus of the renal cortex without crossreaction to the uriniferous tubules (Fig. 3). The reaction
intensity for the glomerulus of PMab-1 is similar to that of
Mab-8.1.1, suggesting that PMab-1 is a useful antibody tomouse podoplanin as well as Mab-8.1.1. In the immunostaining for podoplanin of mouse lungs, the pulmonary
pleura and a part of the pulmonary alveoli were immunostained by PMab-1 and Mab-8.1.1 (Fig. 3). The immunoreaction of Mab-8.1.1 to alveolar cells was stronger andbroader than that of PMab-1. In the immunostaining for
α-SMA and podoplanin of mouse tongues, anti-α-SMA
reacted to blood vessels, whereas PMab-1 reacted to lymphatic vessels in the tunica muscularis, indicating that
PMab-1 is also useful to discriminate lymphatic vessels
from blood vessels in mouse somatic tissue (Fig. 4).
Immunohistochemical studies have recently demonstrated that podoplanin is a salivary gland myoepithelial cell
antigen [4, 5, 20]. In this study of mouse lingual glands,
PMab-1 reacted to acini, duct, and lymphatic vessels without cross-reaction to other tissue, while anti-α-SMA reacted
to acini and ducts with cross-reaction to intra-acinar components and lingual mucous epithelia (Fig. 4). Further, inthe mouse sublingual glands, the regions of myoepithelial
cells around acini (Fig. 5) were immunostained by anti-αSMA, Mab-8.1.1, and PMab-1. The Mab-8.1.1 reacted
more strongly to the PMab-1-reacting region but also
showed a weak cross-reaction with connective tissue
around the striated duct, suggesting that PMab-1 could bea useful antibody to identify podoplanin on salivary gland
myoepithelial cells. The cell-cell junction of acinar cells andthe luminal side of the intercalated duct, where myoepithelial cells are absent, were immunostained by anti-α-SMA
but not by Mab-8.1.1 or PMab-1, suggesting that Mab-8.1.1
and PMab-1 are superior to anti-α-SMA in identifying
myoepithelial cells.
In human sublingual glands, α-SMA-positive myoepithelial cells around mucous acini were immunostainedby the anti-human podoplanin antibodies NZ-1.2 and AB3,
while the basal portion of serous acini partially showed aweak reaction to the antibodies (Fig. 6). Myoepithelial cell
shape varies considerably with the different glands in relation to the physical properties of secretions. In the parotid
gland few myoepithelial cells are on the terminal portion ofserous acini while in the sublingual gland, approximately
50% of the terminal portion surface was covered by myoepithelial cells with a number of broad and extensive
cellular processes on the mucous acini, and with a few

236

Kaji et al.

thin processes on the serous acini [19]. These coincidewith the immunostaining of acini of sublingual gland by
NZ-1.2, suggesting the specificity of NZ-1.2 for human
podoplanin on the myoepithelial cells. The cell-cell junctions of acinar cells where myoepithelial cells are absent
were also immunostained by anti-α-SMA but not by AB3
or NZ-1.2. The NZ-1.2 reacted to a very similar region asthat stained by AB3 and to a much higher extent, and ABS
showed a cross-reaction with nuclei, suggesting that NZ-1.2
is more sensitive and highly specific against the podoplaninof human myoepithelial cells when compared with AB3.
The immunostaining of paraffin-embedded sections
showed a reaction with PMab-1 to the mouse kidney
glomerulus, pulmonary alveoli, and lung lymphatic vessels
without any cross reactions (Fig. 7). Further, the immunostaining of paraffin-embedded sections showed the reactionof NZ-1.2 to monkey glomerulus and kidney lymphatic
vessels, and to monkey pulmonary alveoli and lung lymphatic vessels. The epitope of NZ-1.2 is the amino acids
38–51 of the human podoplanin (Accession No. AB127958)
which is 100% homologous to amino acids 38–51 of themonkey podoplanin (Accession No. XM_001106933.2.).
These suggest that PMab-1 and NZ-1.2 are useful for the
immunostaining for podoplanin on paraffin-embedded sections of mouse and human/monkey as well as on frozen
sections.
In conclusion, rat monoclonal antibodies PMab-1 tomouse podoplanin and NZ-1.2 to human podoplanin were
established. These antibodies were shown to be useful forboth immunostaining and Western-blot.

4.

5.

6.

7.

8.

9.

10.

11.

12.

V.

Acknowledgments

We thank Ryo Yanagiya, Shunpei Morita, Imi Saito,
and Junko Aita for their excellent technical assistance. This
work was supported in part by Grant-in-Aid for Young
Scientists (B) 23701043 and 23791584 (Kaneko and Kato),
Scientific Research (B) 22390345 (principal: Sawa), andExploratory Research 23659884 (principal: Sawa) fromJapan Society for the Promotion of Science, by Mitsubishi
Pharma Research Foundation (Kato and Kaneko), by Children Cancer Association of Japan (Kato), by Office for
Gender Equality of Yamagata University (Kaneko), and byIntelligent Cosmos Academic Foundation (Kato).

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