Journal of Histochemistry
& Cytochemistry
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The Sodium Dependent Inorganic Phosphate Transporter SLC34A1 (NaPi-IIa) is not Localised in the Mouse
Brain: A Case of Tissue-Specific Antigenic Cross-Reactivity
Max Larsson, Cecilie Morland, Irais Poblete-Naredo, Jürg Biber, Niels Christian Danbolt and Vidar Gundersen
J Histochem Cytochem published online 23 May 2011
DOI: 10.1369/0022155411411713
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JHC Express
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This article can be cited as DOI: 10.1369/0022155411411713
The Sodium Dependent Inorganic Phosphate Transporter SLC34A1 (NaPi-IIa) is not
Localised in the Mouse Brain: A Case of Tissue-Specific Antigenic Cross-Reactivity
Max Larsson1,2, Cecilie Morland1, Irais Poblete-Naredo3, Jürg Biber4, Niels Christian Danbolt1,
Vidar Gundersen1,5*
1
Department of Anatomy and Centre for Molecular Biology and Neuroscience, University of
Oslo, P.O. Box 1105 Blindern, N-0317 Oslo, Norway. 2Department of Neuroscience, Karolinska
Institutet, Retzius väg 8, S-171 77 Stockholm, Sweden. 3 Departamento de Genética y Biología
Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional,
Apartado Postal 14-740, México D.F. Mexico. 4Institute of Physiology, University Zürich,
Winterthurerstrasse 190, 8057 Zürich, Switzerland. 5Department of Neurology, Oslo University
Hospital, Norway
*corresponding author: Vidar Gundersen, Department of Anatomy and Centre for Molecular
Biology and Neuroscience, University of Oslo, P.O. Box 1105 Blindern, N-0317 Oslo, Norway,
vidar.gundersen@medisin.uio.no
Received for publication February 9, 2011; accepted May 5, 2011
Short Title:
PHOSPHATE TRANSPORTER ANTIBODIES AND BRAIN CROSS-REACTIVITY
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ABSTRACT
The sodium dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here
we used a polyclonal antiserum raised against NaPi-IIa and NaPi-IIa deficient mice to
characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa
immunoreactive band (~90 kDa) was only present in wild type kidney membranes, and not in
kidney knockout or wild type brain membranes. In the water soluble fraction of wild-type and
knock-out brains another band (~50 kDa) was observed; this band was not detected in kidney.
Light- and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed
immunolabeling of kidney tubules in wild-type, but not knock-out mice. In the brain labeling of
presynaptic nerve terminals was present also in NaPi-IIa deficient mice. This labeling pattern
was also produced by the NaPi-IIa pre-immune serum. We conclude that the polyclonal
antiserum is specific towards NaPi-IIa in the kidney, but in the brain immunolabeling is caused
by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the
kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating
antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest
and underlines the utility of specificity testing using pre-immune sera.
Key words: electron microscopy, immunofluorescence, polyreactive, polyclonal, specificity
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The inorganic Na+-dependent phosphate co-transporter NaPi-IIa belongs to the solute carrier
family 34 (SLC34) (Murer et al., 2004). NaPi-IIa was originally cloned from rat kidney cortex
(Magagnin et al., 1993), and immunocytochemical examination showed that it was located in
microvilli of the brush border membrane of proximal tubules (Custer et al., 1994). Although
NaPi-IIa mRNA expression was initially only found in the kidney, and not in for instance the
brain (Magagnin et al., 1993), NaPi-IIa expression at the protein and mRNA levels was later
detected by immunohistochemistry and RT-PCR in the brain (Hisano et al., 1997). We
hypothesized that NaPi-IIa may be localized to synaptic vesicles, where it, in congruence with
other Na+/phosphate transporters such as the vesicular glutamate transporters, could function as a
vesicular transporter of organic molecules. Indeed, in initial immunoelectron microscopic
experiments using an antibody known to specifically recognize NaPi-IIa in the kidney (Custer et
al., 1994), we detected NaPi-IIa immunolabeling over synaptic vesicle clusters in the rat
hippocampus. To verify the specificity of this labeling, we examined tissue from NaPi-IIa
knockout and wild-type brain and kidney. We describe some of the difficulties in determining
the specificity of an antibody using tissue from knockout animals.
MATERIALS AND METHODS
Animals and tissue preparation
Slc34a1-/- mice, kindly provided by HS Tenenhouse, was generated by deleting exons VI-X of
the slc34A1 gene [described in detail in Beck et al. (1998)]. For light and electron microscopy,
adult knockout mice and wild-type littermates were anesthetized with sodium pentobarbital (50
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mg/kg, i.p.) and fixed by transcardial perfusion with 4 % paraformaldehyde in 0.1 M phosphate
buffer (pH 7.4). The brains and kidneys were removed and placed in phosphate buffer. For light
microscopic immunohistochemistry, the tissue was cryoprotected in 30 % sucrose and cut in 40
µm-thick parasagittal (brain) or horizontal (kidney) sections and processed for
immunofluorescence as described below. For immunoelectron microscopy, tissue specimens of
the hippocampal CA1 region and renal cortex were dissected and embedded in Lowicryl HM20
resin as described (Bergersen et al., 2008). Ultrathin sections were cut from the embedded tissue,
placed on Ni mesh grids and subjected to postembedding immunogold labeling as described
below. For western blotting, after decapitation the brains and kidneys of knock-out and wild-type
mice were quickly dissected, immediately frozen in liquid nitrogen and stored at -80 ˚C. Animal
experiments were performed in accordance with the guidelines of the Norwegian Committees on
Animal Experimentation (Norwegian Animal Welfare Act and European Communities Council
Directive of 24 November 1986-86/609/EEC).
NaPi-IIa antiserum
The polyclonal antiserum against the NaPi-IIa protein was raised in rabbit against a peptide
(MMSYSERLGGPAV) derived from the N-terminal region of the rat NaPi-IIa protein (Custer et
al., 1994). The antibody labels the luminal membrane of renal proximal tubules by
immunofluorescence, and by Western blotting it detects a band of ~90 kDa in the renal brush
border membrane fraction obtained from rat or wild-type mice (Custer et al., 1994, Beck et al.,
1998). This band is absent in mice lacking NaPi-IIa (Beck et al., 1998). NaP-IIa pre-immune
serum was obtained from the rabbits before immunisation with the NaPi-IIa peptide.
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Immunofluorescence
Free floating tissue sections were sequentially incubated in blocking buffer [phosphate buffered
saline (PBS) with 3 % normal goat serum, 0.5 % bovine serum albumin and 0.5 % Triton X-100]
for 2 h, anti-NaPi-IIa 1:1000 in blocking buffer overnight at room temperature, PBS 6 × 10 min,
goat anti-rabbit Alexa Fluor 488 (Invitrogen) 1:1000 in blocking buffer for 2 h, and PBS 3 × 10
min. After mounting, sections were covered with Prolong Gold (Invitrogen) and examined using
the epifluorescence mode of a Zeiss LSM 5 confocal microscope. For immunofluorescence
microscopy one pair of NaPi-IIa knockout and wildtype mice was analysed.
Postembedding immunogold labeling
Ultrathin sections of hippocampal and renal cortex tissue were subjected to postembedding
immunogold labeling essentially as described (Bergersen et al., 2008). In brief, sections were
incubated in 50 mM glycine in Tris-buffered saline (0.3 % NaCl, pH 7.4) with 0.1 % Triton X100 (TBST) for 10 min, followed by blocking buffer (TBST supplemented with 2 % human
serum albumin) for 10 min and primary antibody solution (anti-NaPi-IIa 1:300 or NaP-IIa preimmune serum 1:300 in blocking buffer) overnight at 4°C. After rinsing in TBST, the sections
were incubated in blocking buffer for 10 min and for 1 h in goat anti-rabbit coupled to 10 nm
colloidal gold (British Biocell, Cardiff, UK) diluted 1:20 in blocking buffer. The sections were
rinsed in H2O and counterstained with uranyl acetate and lead citrate before examination in a
Tecnai 12 electron microscope. For immunogold electron microscopy another pair of NaPi-IIa
knockout and wildtype than that used for immunofluorescence microscopy was used.
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Western blotting
Brain and kidney tissue was homogenized in water containing 5 mM EDTA and 1mM PMSF
(freshly dissolved in DMSO). Then the homogenate was centrifuged at 39000 × g (18000 rpm)
for 10 min at 4˚ C, before the pellet was dissolved in SDS-solubilisation buffer (1 % SDS with
150 mM NaCl and 10 mM NaPi pH 7.4), to which 1 mM PMSF was added. This mixture was
incubated for 10 min at room temperature and centrifuged at 1000 × g for 10 min at 15˚ C to
remove unsolubilized material. The water-soluble supernatant fraction and SDS-soluble pellet
fraction were subjected to SDS-PAGE and Western blotting according to standard protocol. The
blots were incubated overnight at 4°C in anti-NaPi-IIa (final dilution 1:3000) in Tris-buffered
saline (pH 7.4) with 1 % BSA and 0.05 % Tween 20. After rinsings, the blots were incubated in
secondary anti-rabbit IgG peroxidase conjugated antibodies (Chemicon, dilution 1:10000) in
Tris-buffered saline (pH 7.4) with 4% dry milk powder 0.05 % Tween 20. The blots were
developed with SuperSignal West Dura Extended Duration Substrate, and analysed in a Fuji
LAS-3000 luminescence scanner. The Western blots were made from homogenates of 3 NaPi-IIa
knockout and 3 wildtype mice. The Western blots were performed twice and gave similar results
in both instances.
RESULTS
Immunocytochemistry
Tissue sections were immunofluorescence labelled with the NaPi-IIa antiserum and examined by
epifluorescence microscopy. The luminal part of kidney proximal tubules of wild-type mice were
strongly immunolabeled in accordance with previous reports (Custer et al., 1994), whereas NaPiCopyright © 2011 The Author(s)
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IIa deficient mice showed no such labeling (Fig. 1A, B). Immunofluorescently stained brain
sections from wild-type mice showed scattered labelled neuronal cell bodies and a dotted
neuropil staining. However, the immunolabeling pattern observed in brain tissue obtained from
knock-out mice was indistinguishable from that of the wild-type (Fig. 1C, D). Postembedding
immunogold labeling and electron microscopy were used to characterize the distribution of
immunolabeling at the ultrastructural level. In ultrathin sections of kidney cortex from wild-type
mice, NaPi-IIa immunolabeling was predominantly situated in the apical microvilli of proximal
tubules (Fig. 2A), whereas only faint immunolabeling was observed in the proximal tubular
microvilli of mice lacking NaPi-IIa (Fig. 2B). In the CA1 region of the hippocampus, a
conspicuous immunolabeling over presynaptic nerve terminals, apparently associated with
synaptic vesicle clusters, was found both in knockout and wild type mice (Fig. 2C-D). However,
this labeling pattern was also produced by the NaPi-IIa pre-immune serum (Fig. 2E and F).
Immunoblotting
Next, we made Western blots of membrane and cytosolic fractions of brain and kidney
homogenates using the NaPi-IIa antiserum. In wild type kidney we detected a single strong band
at about 90 kDa in the SDS-soluble membrane fraction, but no substantial bands were evident in
the cytosolic (water soluble) fraction (Fig. 3). This was as expected considering that NaPi-IIa is a
membrane protein. No bands were observed in either membrane or cytosolic fractions from
kidney of NaPi-IIa knockout mice. However, in whole brain homogenates a distinct band at
about 50 kDa was present in the cytosolic fraction, but not in the membrane fraction in both
NaPi-IIa knockout and wildtype mice (Fig. 3). Notably, this 50 kDa band was not detected in
water-soluble kidney fraction and only very faintly so (compared to the major band at 90 kDa) in
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the membrane fraction of wild-type and knock-out kidney homogenate. As this band was
present also in knockout tissue, it is unlikely to represent a breakdown product of the full length
NaPi-IIa protein, but rather corresponds to an unrelated, cytosolic protein for which the
antiserum is specific. Importantly, this second protein appears to not be expressed to an
appreciable extent in kidney, resulting in immunolabeling that is highly specific for NaPi-IIa in
this tissue.
DISCUSSION
The original aim of this study was to investigate whether the Na+/inorganic phosphate transporter
NaPi-IIa, which is essential for retention of inorganic phosphate in the kidney (Beck et al.,
1998), is present in synaptic vesicles in the central nervous system, where, we hypothesized, it
could function to transport organic ions over the vesicle membrane. Initial support for this
hypothesis was obtained by immunohistochemical labeling of NaPi-IIa in rat (not shown) and
wildtype mice, which showed punctate staining at the light microscopic level and presynaptic
labeling that appeared associated with synaptic vesicles at the electron microscopic level.
Furthermore, the polyclonal NaPi-IIa antiserum used produced no detectable immunofluorescent
staining and only faint postembedding immunogold labeling in kidney sections from mice
lacking NaPi-IIa. However, when attempting to corroborate the specificity of the
immunohistochemical reaction in the brain using nervous tissue from knockout animals, neither
the distribution nor strength of immunolabeling differed from that observed in brain tissue of
wild-type mice. Thus, the antiserum specifically detects NaPi-IIa in the kidney but not in the
brain.
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The failure to detect NaPi-IIa in the brain in the present study is consistent with the study by
Magagnin et al. (1993), who did not detect NaPi-IIa mRNA by Northern blotting in brain tissue,
but appears at odds with the work of Hisano and colleagues (1997) who used the more sensitive
RT-PCR technique and reported NaPi-IIa expression in the rat brain. Using an antibody directed
to the C-terminus of NaPi-IIa, they further concluded by immunocytochemistry that NaPi-IIa is
confined to subsets of neuronal somata and fibers and of ependymal and endothelial cells
(Hisano et al., 1997). However, because NaPi-IIa deficient animals were not available, the
immunocytochemical signal could not be validated. Here we have used an antibody raised
against the N-terminal part of NaPi-IIa that produced a quite different labeling pattern in the
brain. Although a weak NaPi-IIa expression may have been masked by a stronger expression of
the unrelated cross-reacting protein in our immunohistochemical experiments, our inability to
detect NaPi-IIa in the brain also by Western blotting suggest that this transporter is not
expressed, or only very weakly so, in the mouse brain.
It should be noted in parallel experiments of rat brain tissue (not shown) the NaPi-IIa
antiserum produced a 50 kDa band on Western blots of whole brain homogenates and isolated
synaptic vesicles, as well as the same hippocampal immunogold labeling as that observed in the
mouse. Thus, we think that the cross-reacting protein is present across different species.
The present data show that antibodies specific and selective for a particular protein in one
type of tissue are not necessarily so in other tissue types, underlining the importance that
specificity testing of antibodies should be performed in the tissue that is the subject of the study.
Therefore, using as a specificity control system cell cultures transfected for the protein of interest
is not sufficient to rule out cross-reactivity. Antibodies should, whenever possible, be tested, in
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knockout tissue that is as identical as possible to that under immunohistochemical investigation
(Holmseth et al., 2006).
When animals are injected with an antigen, multiple B-cells are usually activated
resulting in a number of different cell clones producing different amounts of antibodies to the
same antigen. The desired antibodies are of course those that only recognize the antigen of
interest, and a good antiserum contains high levels of these. However, antibodies recognizing
also other molecules are usually produced. Some of these antibody clones are polyreactive, i.e.
they are directed to several distinct epitopes (Kramer et al., 1997, Danbolt et al., 1998), often
with high-affinity against one epitope and lower affinities against others (Mouquet et al., 2010).
This means that also monoclonal antibodies can produce cross-reactive immunohistochemical
labeling, like that observed in this study. Thus, specificity testing should be just as rigorous for
monoclonal as for polyclonal antibodies.
Another question is whether we could have discovered that the NaPi-IIa labeling of brain
tissue was unspecific without using the NaPi-IIa knock out tissue. In wildtype and NaPi-IIa
knockout hippocampus the labeling pattern produced by the pre-immune serum and the NaPi-II
antiserum was similar. This is strong evidence that the cross-reacting antibodies were present in
the serum before the immunisation procedure with the specific NaPi-IIa antigen, and underscores
the usefulness of performing controls with pre-immune sera. Peptide absorption controls can
also give certain indications about antibody specificity. If a peptide used for producing an
antiserum does not block the immunolabeling, this may indicate that the cross-reacting
antibodies are not those that bind to the desired antigen (which,given the labeling produced by
the preimmune serum, is presumably the case here). If, however, the antigenic peptide blocks the
labeling, this does not necessarily mean that the labeling is specific for the transporter, because
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the cross-reaction could be produced by polyreacting antibodies (see Holmseth et al., 2005,
2006). In sum, the present results highlight the importance of performing appropriate specificity
controls of antibody reactivities (Saper and Sawchenko, 2003, Holmseth et al., 2005, Holmseth
et al., 2006). The NaPi-IIa antibodies are an example of a situation where antibodies specific for
a protein in one tissue is not selectively specific for that protein in another tissue. The best way
of ascertaining that an immunohistochemical signal in a given tissue results from specific
antibody binding of the antigen of interest is by observing that this signal is absent in the same
tissue lacking the antigen.
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Figure 1. (A, B) Immunofluorescent staining of NaPi-IIa in kidney proximal tubules from wildtype (A, WT) and NaPi-IIa deficient (B, KO) mice. (C, D) NaPi-IIa immunofluorescence
labeling in cerebral cortex from wild-type (C) and NaPi-IIa deficient (D) mice. Insets show
punctate staining of neuropil. Arrowheads indicate immunolabeled cell bodies. Scale bar in D, 20
µm, valid for all panels.
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Figure 2. (A, B) Postembedding immunogold labeling of NaPi-IIa in kidney proximal tubules
from wild-type (A, WT) and NaPi-IIa deficient (B, KO) mice. (C-F) Examples of immunogold
labeling produced by the anti-NaPi-IIa antibodies (C, D) and the NaPi-IIa pre-immune serum (E,
F). Note that presynaptic terminals (t) forming asymmetric synapses with dendritic spines (s) in
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the stratum radiatum of hippocampal CA1 from wild-type (C, E) and NaPi-IIa deficient (D, F)
mice are labelled. Scale bar in F, 200 nm, valid for all panels.
Figure 3. Western blot of NaPi-IIa of SDS-soluble membrane fractions and water-soluble
cytosolic fractions of kidney (K) and brain (B) homogenate obtained from wild-type (WT) and
NaPi-IIa deficient (KO) mice. Arrow indicates the NaPi-IIa reactive band.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the authorship and publication of
this article.
Funding
This work was funded by grants from the Research Council of Norway, including from FUGE
(183727; NCD).
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