Histochem Cell Biol (2005) 123: 639–646
DOI 10.1007/s00418-005-0791-4

O R I GI N A L P A P E R

Akihiro Hosoya Æ Kazuto Hoshi Æ Noriyuki Sahara
Tadashi Ninomiya Æ Syoji Akahane
Tadafumi Kawamoto Æ Hidehiro Ozawa

Effects of fixation and decalcification on the immunohistochemical
localization of bone matrix proteins in fresh-frozen bone sections
Accepted: 1 March 2005 / Published online: 7 June 2005
Ó Springer-Verlag 2005

Abstract To examine the stability of bone matrix proteins for crystal dislocation, the immunolocalization oftype I collagen, bone sialoprotein, and osteopontin wasinvestigated during diﬀerent stages of ﬁxation anddecalciﬁcation. Four-week-old rat femurs were rapidly
frozen, and were sectioned without ﬁxation or decalciﬁcation. Thereafter, following or bypassing ﬁxation in
4% paraformaldehyde, these sections were decalciﬁed in
5% EDTA for 0-5 min. Before decalciﬁcation, marked
radiopacity of bone matrix was observed in contact
microradiography (CMR) images, and electron probe
microanalysis (EPMA) demonstrated intense localization for phosphorus and calcium. In ﬁxed and unﬁxed
sections without decalciﬁcation, immunolocalization ofbone matrix proteins were almost restricted to osteoid.
After 1 min of decalciﬁcation, reduced radiopacity wasapparent in the CMR images, and less phosphorus andcalcium was observed by EPMA, which completely
disappeared by 5 min decalciﬁcation. After 3-5 min of
decalciﬁcation, unﬁxed sections showed that these proteins were immunolocalized in bone matrix, but were notdetectable in osteoid. However, ﬁxed sections demonstrated that these were found in both bone matrix andA. Hosoya (&) Æ N. Sahara Æ H. Ozawa
Department of Oral Histology,
Matsumoto Dental University,
1780 Gobara Hirooka, Shiojiri,
Nagano 399-0781, Japan
E-mail: hosoya@po.mdu.ac.jp
Tel.: +81-263-51-2043
Fax: +81-263-53-3456
K. Hoshi
Department of Menicon Cartilage and Bone Regeneration,
Graduate School of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
N. Sahara Æ T. Ninomiya Æ S. Akahane Æ H. Ozawa
Institute for Dental Science, Matsumoto Dental University,
1780 Gobara Hirooka, Shiojiri, Nagano 399-0781, Japan
T. Kawamoto
RI Research Institute, School of Dental Medicine,
Tsurumi University, 2-1-3 Tsurumi,
Tsurumi-ku, Yokohama, Kanagawa 230-8501, Japan

osteoid. The present ﬁndings suggest that bone matrix
proteins are embedded in calciﬁed matrix which is separated from the aqueous environment and that theyhardly move, probably due to ﬁrm bonding with eachother. In contrast, matrix proteins in osteoid are subject
to loss after decalciﬁcation because they may be boundto scattered apatite crystals, not to each other.
Keywords Fresh-frozen section Æ Non-decalciﬁed
section Æ Bone matrix proteins Æ
Immunohistochemistry Æ Bone

Introduction
Histochemical methods have progressed well to date,because the exact protein localization or gene expression
can be detected using enzyme and immuno-cytohistochemistry or in situ hybridization. However, calciﬁed
hard tissue, such as bone and teeth, remains diﬃcult toanalyze due to the hardness of tissue samples. Althoughboth ﬁxation and decalciﬁcation are usually needed forconventional histological and histochemical observations, these procedures may cause denaturation of proteins, thus resulting in a reduction in enzyme activity andimmunoreactivity (Baker et al. 1958; Takeshita et al.
1983; Mullink et al. 1985; Mukai et al. 1986; Van
Noorden and Vogels 1986; Wakisaka 1986; Van Noorden et al. 1989; Van Den Munckhof et al. 1994; Fukase1997). In particular, decalciﬁcation may aﬀect the degreeof staining, since some materials are easily lost and/or
dislocated from the original sites during the process. In
order to avoid the disadvantage associated with ﬁxationand decalciﬁcation, a number of attempts have been
made to obtain unﬁxed and non-decalciﬁed sections ofcalciﬁed tissue (Hammarstrom 1986; Hill and Elde 1990;
Fink 1992; McElroy et al. 1993; Shimada and Watanabe
1995). However, most of those previous sections could
be improved, because the sections were too thick andbecause the sizes of the available samples were limited to640small ones. Therefore, many researchers have tried toestablish new techniques that enable whole bone or
tooth sectioning without ﬁxation or decalciﬁcation.
In 2000, a new method of fresh-frozen sectioning of
calciﬁed hard tissue was reported by Kawamoto andShimizu (2000). It provides less than 2 lm thick sectionsof calciﬁed tissues without ﬁxation and decalciﬁcation,and is widely applicable for various morphologicalanalyses such as histochemistry, immunohistochemistry,in situ hybridization, and laser microdisectioning (Kawamoto 2003). This method has several advantages, inthat sections can be prepared from their original specimens within several hours, while loss and/or dislocationof water-soluble materials is prevented, since the samples do not undergo chemical ﬁxation and decalciﬁcation, thus preserving the immunoreactivity of eachsection.
This method can be used to detect the exact immunohistochemical localization of various calciﬁed tissue
proteins. Calciﬁed hard tissue contains type I collagen,as well as several non-collagenous proteins, such as bonesialoprotein (BSP) and osteopontin (OPN). Type I collagen localized not only the calciﬁed matrix or theirformative cells, but also a wide range of connective tissues, such as skin, muscle, periosteum, and tendon(Nanci 2003). BSP, on the other hand, is conﬁned tobone, dentin, cementum, and cartilage, as well as thecells which produce these tissues (Begue-Kirn et al. 1994;
Macneil et al. 1994; Bosshardt and Nanci 1998). OPN is
more widely distributed than BSP (Helder et al. 1993;
Bronckers et al. 1994). In addition to areas of BSP
localization, OPN is present in non-mineralized tissue,
such as kidney (Mark et al. 1988) and urinary tractepithelium (Arafat et al. 2002), as well as in macrophages (Miyazaki et al. 1990), and activated T-lymphocytes (Patarca et al. 1986). All of these collagen andsialic acid-rich proteins seem to be associated with themineralization process (Hunter et al. 1996; Hoshi et al.
2001), but the interactions among these proteins andminerals should be elucidated in more detail. Thus, inthe present experiment, we used intact calciﬁed tissue
sections without ﬁxation and decalciﬁcation, treated thesections with various degrees of ﬁxation and decalciﬁcation, and examined the immunolocalization of calciﬁed tissue proteins in those sections. Therefore, thismethod is the ﬁrst technique that can reveal bonding
condition among bone matrix proteins and minerals in
calciﬁed tissue sections.

Materials and methods
Preparation of frozen and non-decalciﬁed sections
All experiments were performed according to strict
guidelines set forth by the Matsumoto Dental University
Committee on Intramural Animal Use. Four-week-old
Wistar rat (weighing 85–90 g) femurs were used.
Following anesthesia (intraperitoneal injection ofpentobarbital, 40 mg/kg), the femurs were removed and
immediately frozen in hexane ( 80°C), cooled with acooling apparatus (PSL-1800; Tokyo Rikakikai Co. Ltd,
Japan). Each sample was embedded in 5% carboxymethyl cellulose (CMC) gel and completely frozen. Eachfrozen CMC sample was then attached to the sample
stage of a cryomicrotome (CM3500; Leica Instruments,
Germany) in a cryochamber ( 25°C). After sitting for
30 min, each CMC sample was covered with a polyvinylidene chloride ﬁlm (10 lm thick; Asahikasei Kogyo
Co., Japan), coated with synthetic rubber cement(Cryoglue Type I; FINETEC Co. Ltd, Japan), andsagittally sectioned with the ﬁlm, at a thickness of 5 lm,
using a disposable tungsten carbide blade (Jung TC-65;
Leica Instruments, Germany).
Contact microradiogram observations
Mineral density was examined by contact microradiography (CMR). Sections were removed from the cryochamber and immersed in 100% ethanol to remove
trapped air bubbles for 5 min, after which they were
ﬁxed with 4% paraformaldehyde in 0.1 M phosphatebuﬀer (pH 7.4) for 5 min at 20°C. After this, the sections
underwent decalciﬁcation with 5% EDTA (pH 7.4) for
0, 1, 3, and 5 min at 20°C. After drying at room temperature, the sections were mounted on a high-resolution ﬁlm (Pelicula So-343; Kodak, Rochester, N.Y.,
USA) and CMR images were taken using a soft X-ray
unit (SOFRON SRO-iM50; SOFRON Co., Japan) at
10.5 kVp, 2 mA, and an exposure time of 6 min.
Electron probe microanalysis (EPMA)
In order to investigate the degree of decalciﬁcation,
phosphorus and calcium were detected by EPMA (JXA8200; JEOL Ltd, Japan). Fixed and decalciﬁed (0 and
1 min) sections were dried, and carbon-coated (JEE-420;
JEOL) to prevent charging. Cortical bone areas were
measured using a beam current of 40 nA and an accelerating voltage of 15 kV.
Histological observations
Fixed and decalciﬁed (0, 1, 3, and 5 min) sections wereused. These sections were stained with 0.1% toluidine
blue (pH 7.0) or 1% alizarin red S for 3 min, after which
they were washed in running water for 5 min. The sections with ﬁlm were placed on the sectioned side on a
glass slide coated with 30% glycerol.
Immunohistochemical observations
For immunohistochemical localizations of type I collagen, BSP, and OPN, ﬁxed and unﬁxed sections were641

treated with 0.3% H2O2 in a solution of 0.01 M phosphate-buﬀered saline (PBS, pH 7.4) for 30 min at room
temperature to incubate endogenous peroxidase. The
sections were then pretreated with 10% bovine serum
albumin (Seikagaku, Tokyo, Japan) in PBS for 30 min
at room temperature and incubated with polyclonal
rabbit antibodies against rat type I collagen (Chemicon,
Temecula, Calif., USA), human BSP (LF-120, kindly
provided by Dr. L.W. Fisher), and mouse OPN (kindly
provided by Dr. M. Fukae) at a 1:100 dilution for 2 h atroom temperature. Sections were then rinsed in PBS andreacted with FITC-conjugated goat anti-rabbit IgG(Chemicon) at a 1:50 dilution for 1 h at room temperature, after which they were visualized using a ﬂuorescence microscope (Axioplan 2; Carl Zeiss, Germany).
Non-immune rabbit serum was diluted to the same
strength for use as negative controls, instead of primary
antibodies.

Results

staining of the bone matrix was observed, and staining
was not evident after 5 min (Fig. 2b-d). Staining of bonemarrow and skeletal muscle was not observed in nondecalciﬁed or decalciﬁed sections.
The CMR images and alizarin red S staining results
suggest that the bone matrix of the cortical area wasdecalciﬁed by about 50% by 3 min, and completely
within 5 min. Trabecular bone showed a similar timecourse of decalciﬁcation (data not shown).
EPMA observations
Phosphorus was distributed extensively throughout
mineralized cortical bone, and slightly in non-mineralized areas of bone marrow and skeletal muscle (Fig. 3a).
After 1 min of decalciﬁcation, almost no phosphorus
was observed in bone and non-mineralized areas(Fig. 3c). Calcium was observed in calciﬁed corticalbone and reduced levels were observed after decalciﬁcation (Fig. 3b, d).

CMR observations
Toluidine blue staining
Calciﬁed bone matrix showed intense radiopacity
without decalciﬁcation (Fig. 1a). After decalciﬁcation,
radiopacity was reduced, and eventually there was noradiopaque area at all by 5 min decalciﬁcation(Fig. 1b–d).
Alizarin red S staining
In the non-decalciﬁed section, almost all bone matrix
was stained strongly, although some areas demonstrated
weaker staining. Osteoid was negative for this staining(Fig. 2a). After decalciﬁcation, a gradual reduction in
Fig. 1 CMR analysis of corticalbone. Sections decalciﬁed for 0(a), 1 (b), 3 (c), and 5 (d) min.
a Calciﬁed bone matrix
demonstrates intenseradiopacity. b Reduced
radiopacity is observed after
decalciﬁcation. c Radiopaque
area is still partially observed.
d There is no radiopaque areaat all. BM bone marrow; M
skeletal muscle. Bar=200 lm

Prior to decalciﬁcation, the bone matrix of cortical bone
did not stain with toluidine blue (Fig. 4a). After decalciﬁcation, however, a gradual increase in metachromagia with toluidine blue staining was observed. The
intensity of staining of bone marrow and skeletal muscle
did not change with decalciﬁcation (Fig. 4b–d). In trabecular bone, bone matrix was hardly stained without
decalciﬁcation (Fig. 4e). After 1 min of decalciﬁcation,
the cartilage matrix embedded in the central part oftrabecular bone was metachromatic, while osteoid in thesurface of trabecular bone did not show the metachromagia (Fig. 4f). After 3 min, the metachromagia of the642
Fig. 2 Alizarin red S staining ofcortical bone. Sections
decalciﬁed for 0 (a), 1(b), 3(c), and 5 (d) min. a Intense
staining is apparent throughout
the bone matrix, although none
was observed in osteoid (arrowhead). b–d Following
decalciﬁcation, a gradualreduction in intensity ofstaining of bone matrix isobserved, which disappeared
after 5 min of decalciﬁcation(b–d). BM bone marrow; CB
cortical bone; M skeletal
muscle. Bar=200 lm

Fig. 3 Phosphorus (a, c) andcalcium (b, d) mapping images
using EPMA after 0 (a, b) and 1(c, d) min of decalciﬁcation.
a Phosphorus is localized
intensively in cortical bone, andslightly in non-mineralized
areas. b Calcium is detected incalciﬁed bone. c-d Following
decalciﬁcation, phosphorus andcalcium localizations are clearlydecreased. Bar=100 lm

calciﬁed cartilage had faded, and it completely disappeared after 5 min (Fig. 4g–h).
Immunohistochemical observations
The patterns of localization of type 1 collagen and two
non-collagenous proteins (BSP and OPN) are summarized in Tables 1 and 2, respectively.
Unﬁxed sections
Prior to decalciﬁcation, immunoreactivity for type 1
collagen was observed at the periphery of trabecularbone corresponding to osteoid, and reticulately in bonemarrow. In contrast, bone matrix was hardly stained(Fig. 5a). Following decalciﬁcation, increased immunostaining for type I collagen in the bone matrix was observed, although its localization diminished at the
periphery of bone (Fig. 5b, c). The stainings in bonemarrow were observed reticulately in decalciﬁed as well
as non-decalciﬁed sections (Fig. 5 a–c).
Intense immunoreactivity for BSP and OPN was
observed in the periphery of trabecular bone, but not inbone matrix of non-decalciﬁed sections. (Fig. 5d, g).
After decalciﬁcation, immunoreaction of BSP and OPN
was strongly observed lineally in bone matrix, aroundthe cement line. The central part of trabecular bone,corresponding to the calciﬁed cartilage matrix, was not
immunoreactive for BSP or OPN. Immunoreactivity for
BSP and OPN appeared in bone matrix and disappeared

643
Fig. 4 Toluidine blue stainingof cortical (a–d) and trabecularbone (e–i). Sections decalciﬁedfor 0 (a, e), 1(b, f), 3 (c, g), and 5(d, h) min. a, e Toluidine blue
staining is faint in cortical (CB)and trabecular bone prior to
decalciﬁcation. b–d A gradualincrease in metachromagia ofcalciﬁed cortical bone matrix
was observed as decalciﬁcation
progressed. f–h Intense
metachromagia of calciﬁed
cartilage matrix of trabecularbone (arrows in f) is observed
after 1 min of decalciﬁcation.
Osteoid (arrow head) is hardly
stained regardless of the degreeof decalciﬁcation. As
decalciﬁcation progressed, areduction in metachromagia ofcalciﬁed cartilage matrix wasseen. BM bone marrow; C
cartilage; CB cortical bone;
M skeletal muscle; Ob
odontoblast. Bar=200 lm(a–d), 100 lm (e–h)

from the periphery of bone after decalciﬁcation (Fig. 5e,f, h, i). OPN demonstrated a similar pattern of immunoreactivity as BSP; however, weaker staining of thebone matrix was observed (Fig. 5h).

None of the speciﬁc localizations was identiﬁed in thenegative controls for type I collagen, BSP or OPN (datanot shown).

Discussion
Fixed sections
Type I collagen was localized at the periphery of trabecular bone and in bone marrow as in unﬁxed and nondecalciﬁed sections (Fig. 6a). After decalciﬁcation, degree of immunostaining for type I collagen increased incalciﬁed bone matrix, and staining at the periphery ofbone remained (Fig. 6b–c).
In non-decalciﬁed sections, intense immunostaining
for BSP and OPN was observed at the periphery ofbone, while almost none was observed in calciﬁed bonematrix (Fig. 6d, g). After decalciﬁcation, intense staining
for BSP and OPN was observed in areas corresponding
to osteoid and cement lines, as well as in bone matrix(Fig. 6e, f, h, i). OPN showed the same localization as
BSP, but the staining in bone matrix was weaker thanthat of BSP (Fig. 6h–i). Calciﬁed cartilage matrix
showed no immunoreactivity for BSP or OPN(Fig. 6a–i).

A number of strategies have been devised to enable
researchers to examine calciﬁed hard tissue without
decalciﬁcation. The method of fresh-frozen sectioning,ﬁrst described by Kawamoto and Shimizu (2000), is
useful for the preparation of the serious unﬁxed andnon-decalciﬁed sections. In the present study, we
examined 5 lm thick fresh-frozen bone sections. Thesesections were decalciﬁed by about 50% within 3 min,and completely by 5 min using 5% EDTA, in CMR
images and alizarin red S staining results. Further,
EPMA results showed a reduction in phosphorus andcalcium content after 1 min of decalciﬁcation. Using
these sections, we analyzed immunohistochemically the
relation between the stability of bone matrix proteinsand crystal dislocation.
In unﬁxed sections without decalciﬁcation, immunoreactivity for type I collagen, BSP, and OPN was restricted to osteoid. After decalciﬁcation, however,644
Table 1 Immunolocalization of type I collagen in trabecular bone
Sections
Non-ﬁxed
Fixed

Bone
Non-decalciﬁed
Decalciﬁed
Non-decalciﬁed
Decalciﬁed+ Immunoreactive,

Osteoid

Cement
line

+

Bone
marrow
+
+
+
+

+
+
+

Negative

Table 2 Immunolocalization of BSP and OPN in trabecular bone
Sections
Non-ﬁxed
Fixed

Bone
Non-decalciﬁed
Decalciﬁed
Non-decalciﬁed
Decalciﬁed+ Immunoreactive,

Osteoid

Cementline

Bonemarrow

+
+
+

+
+
+

+

Negative

immunoreactivity for these proteins was detected inbone matrix, and not in osteoid. It was considered thatthe immunoreactivity of bone matrix proteins might bemasked by minerals, and that after decalciﬁcation, they
become exposed and immunodetectable. Furthermore,
the present ﬁndings suggest that bone matrix proteinsembedded in calciﬁed matrix which is separated from
aqueous environment are relatively immobile, perhaps
because of strong bonds between them. In contrast,
matrix proteins embedded in osteoid might be bound to
scattered apatite crystals, rather than each other, andmay be lost during decalciﬁcation.
Fig. 5 Immunolocalization oftype I collagen (a–c), BSP (d–f),
and OPN (g–i), in trabecularbone. Unﬁxed sections
decalciﬁed for 0 (a, d, g), 3 (b, e,h), and 5 (c, f, i) min. a
Immunolocalization of type I
collagen is observed in the
periphery of trabecular boneaccording to osteoid area(arrow head), as well as in bonemarrow (arrow), although bonematrix is hardly stained.
b–c Type I collagen is found inbone matrix and bone marrow,
but not in osteoid. d, g Staining
for BSP and OPN is restricted
to the osteoid area. e–f, h–i
After decalciﬁcation, increased
immunostaining for BSP and
OPN is observed in the bonematrix, especially the lineal
bone matrix (open arrow), while
diminished staining of osteoid isobserved. Bar=100 lm

Some bone matrix proteins may be cross-linked toeach other, playing roles in bone matrix maturation andcalciﬁcation. OPN forms covalent cross-links withﬁbronectin (Beninati et al. 1994), thereby enhancing its
cell–matrix interfacial adhesion properties and providing
increased physical dimensions. Furthermore, BSP and
OPN in mineralized bone matrix form large protein
aggregates in the presence of tissue transglutaminase(Kaartinen et al. 2002). This further supports ourassertion that BSP and OPN are relatively immobile inmineralized bone following decalciﬁcation. Meanwhile,some proteins, including OPN and BSP, are localized incalciﬁed nodules in osteoid (McKee et al. 1993, Hoshi
et al. 2001). They are thought to associate withhydroxyapatite, and thus to regulate crystal growth andformation. As demonstrated in the present study, since
they may not bind to each other or to other organic
substances, they might be lost or drift when hydroxyapatite disappear after decalciﬁcation. Although BSP
possesses nucleation activity of hydroxyapatite in vitro,
OPN plays a negative role in crystal growth (Hunter
et al. 1996). The ability of those matrix proteins to moveeasily may rather be advantageous for hydroxyapatite
growth and maturation of calciﬁed matrix.
In ﬁxed sections, bone matrix proteins were observedin area of bone matrix and osteoid after decalciﬁcation.
These ﬁndings suggest that ﬁxation with paraformaldehyde, followed by decalciﬁcation with EDTA, preventsloss of bone matrix proteins from osteoid. However, it isworth noting that ﬁxation and decalciﬁcation usually
reduce enzyme activity and immunoreactivity (Baker
et al. 1958; Takeshita et al. 1983; Mullink et al. 1985;

645
Fig. 6 Immunolocalization oftype I collagen (a–c), BSP(d–f), and OPN (g–i), intrabecular bone. Fixed sections
decalciﬁed for 0 (a, d, g), 3(b, e, h) and 5 (c, f, i) min.
a Immunoreaction of type I
collagen is observed in the areaof osteoid (arrow head) andbone marrow (arrow). b-c TypeI collagen localizes in bonematrix, and staining in osteoidarea (arrow) remains.
d, g Immunolocalizations of
BSP and OPN are intensively
found in the osteoid area.
e–f, h–i After decalciﬁcation,these proteins localize in thearea of osteoid, cement line
(open arrow), and bone matrix.
Bar=100 lm.

Mukai et al. 1986; Van Noorden and Vogels 1986;
Wakisaka 1986; Van Noorden et al. 1989; Van Den
Munckhof et al. 1994; Fukase 1997). In addition, reduced ALP activity is noted in fresh-frozen sections,such as those used in the present experiment, following
long-term ﬁxation (24 h) and decalciﬁcation (6 days)(Kawamoto and Shimizu 2000). Because the immunoreactivity of bone matrix proteins, analyzed in thepresent study, was rarely aﬀected by paraformaldehyde
ﬁxation, it could be still observed in the ﬁxed section
after decalciﬁcation.
In non-decalciﬁed sections, immunostaining for bonematrix proteins was observed in osteoid, but hardly inmineralized bone. A number of methods exist for osteoid staining (Tripp and Mackay 1972; Yoshiki 1973;
Villanueva and Mehr 1977); however, they all involvediﬃcult procedures. Our staining methods using nondecalciﬁed fresh-frozen sections can stain to osteoid inserial sections, and can combine with bone labels such as
tetracycline and calcein labeling. Thus, it is thought tobe the useful method for bone morphometry observation.
This study describes a method for fresh-frozen sections, and these observations provided additional insightfor interaction between calciﬁcation and bone matrix.
This method can be prepared by unﬁxed and nondecalciﬁed hard tissue sections, and analyzed for many
types of research including water-soluble molecule distribution and localizations of proteins, which immunoreactivity may reduce by chemical ﬁxation. We believethat this procedure will be become the method of choicefor calciﬁed hard tissue research.

Acknowledgements The authors would like to thank Dr. L.W.
Fisher (National Institute of Dental Research, National Instituteof Health) and Dr. M. Fukae (Department of Biochemistry,
Tsurumi University School of Dental Medicine) for kindly providing the antibodies against BSP and OPN. This work was
supported, in part, by Grant-in-Aid for Scientiﬁc Research from
the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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